Antagonists of interleukin-15

ABSTRACT

Disclosed herein are mutant IL-15 polypeptides and methods for using these polypeptides to modulate the immune response in a patient.

RELATED APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 09/437,585,filed Nov. 9, 1999 (now allowed), which is a continuation-in-part ofU.S. Ser. No. 08/842,947, filed Apr. 25, 1997 (now issued as U.S. Pat.No. 6,001,973), which claims benefit from U.S. Ser. No. 60/016,634,filed Apr. 26, 1996.

FIELD OF THE INVENTION

[0002] The field of the invention is cytokine-mediated therapeutics.

BACKGROUND OF THE INVENTION

[0003] An effective immune response begins when an antigen or mitogentriggers the activation of T cells. In the process of T cell activation,numerous cellular changes occur, which include the expression ofcytokines and cytokine receptors. One of the cytokines involved in theimmune response is interleukin-15 (IL-15). IL-15 is a T cell growthfactor that stimulates the proliferation and differentiation of B cells,T cells, natural killer (NK) cells, and lymphocyte-activated killer(LAK) cells in vitro. In vivo, the proliferation of these cell typesenhances the immune response.

[0004] IL-15 binds to a heterotrimeric receptor that consists of theIL-2Râ subunit, the IL-2Rã subunit, and a unique IL-15Rá subunit.

[0005] Patients benefit from suppression of the immune response in anumber of circumstances, for example, in the event of organtransplantation or autoimmune disease. In other circumstances, forexample when select immune cells have become malignant orautoaggressive, it is beneficial to actively destroy them.

SUMMARY OF THE INVENTION

[0006] The invention features mutants of the cytokine IL-15. Preferably,these mutants bind the IL-15 receptor complex with an affinity similarto wild-type IL-15, but fail to activate signal transduction. The mutantpolypeptides of the invention therefore compete effectively withwild-type IL-15 and, when they do so, they block one or more of theevents that normally occur in response to IL-15 signalling, such ascellular proliferation. By modulating the events mediated by the IL-15receptor complex, mutant IL-15 polypeptides can modulate the immuneresponse, and thus are therapeutically useful.

[0007] Mutant IL-15 polypeptides can have several characteristics thatare advantageous in the context of treatment regimes. First, they areunlikely to be immunogenic because they can differ from wild type IL-15by only a few substituted residues. Second, IL-15 mutants can bindIL-15Rá with the same high affinity as wild type IL-15 and thus, cancompete effectively for the receptor. Third, IL-15 mutants can be easilymodified to remain active in the circulation for a prolonged period oftime, or to produce a lytic response in cells to which they bind. Inaddition, the IL-15 receptor alpha (IL-15Rá) polypeptide is expressed byactivated or malignant immune cells, but not on resting immune cells.Thus, the mutant polypeptide of the invention can be used tospecifically target activated or malignant immune cells.

[0008] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims. Althoughmaterials and methods similar or equivalent to those described hereincan be used in the practice or testing of the invention, the preferredmaterials and methods are described below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0009]FIG. 1 is a photograph of a Western blot depicting thebacterially-produced, affinity-purified FLAG-HMK-IL-15 fusion protein.Proteins were separated by electrophoresis through a 20%SDS-polyacrylamide gel, visualized by staining with Coomassie blue (lanea) and transferred to PVDF membranes. The membranes were exposed toantibodies against the FLAG peptide (lane b) and human IL-15 (lane c).In lane (d), eluates from an anti-FLAG affinity column were incubatedwith heart muscle kinase and [³²P]-ã-ATP for 30 minutes beforeelectrophoresis.

[0010]FIG. 2 is an autoradiograph depicting proteins that were extractedfrom PHA-stimulated PBMCs that were incubated with [³²P]-FLAG-HMK-IL-15,cross-linked with DSS, and separated by SDS-PAGE under reducingconditions. A 75-80 Kda band is apparent (lane 1). To control fornonspecific cross-linking, replicates were incubated with[³²P]-FLAG-HMK-IL-15 in the presence of a molar excess of IL-15 (lane2).

[0011]FIG. 3A is a bar graph depicting the mitogenic response ofPHA-stimulated PBMCs that were stimulated with either buffer alone(“none”), FLAG peptide (10⁻⁴ M), IL-2 (10⁻⁹ M) or FLAG-HMK-IL-15 (10⁻⁹M). The cultured cells were “pulsed” with [³H]-TdR, and the amount ofincorporated radioactivity was determined by scintillation counting.

[0012]FIG. 3B is a bar graph depicting the mitogenic response of BAF-BO3cells transfected with pRcCMV (control; left-hand panel) or withPrccmv-IL-2Râ (encoding the wild-type human IL-2Râ subunit; right-handpanel) and incubated with medium alone (“none”), media containing IL-3,IL-2 (50 U/ml), or FLAG-HMK-IL-15 (10 ng/ml), and pulsed with [³H]-TdR(b). The cultured cells were “pulsed” with [³H]-TdR, and the amount ofincorporated radioactivity was determined by scintillation counting.

[0013] FIGS. 4A-4C are a series of plots depicting the expression ofIL-15Rá subunits on human PBMCs by flow cytometry analysis of stainedcells. Freshly isolated or PHA pre-activated PBMCs were washed andincubated with medium alone (thin line) or FLAG-HMK-IL-15 (thick line)followed by anti-FLAG biotinylated Ab and Streptavidin-RED670 (FIG. 4A).The data presented in FIG. 4B were obtained from washed PBMCs that werepreincubated with media alone (left-hand side) or media containing 2 igof human recombinant IL-15 (right-hand side) for 20 minutes at 4ECbefore the addition of FLAG-HMK-IL-15. For simplicity, graphs representspecific binding of FLAG-HMK-IL-15; non-specific binding was subtracted.The data presented in FIG. 4C were obtained from PBMCs that werepreincubated with phycoerythrin conjugated anti-CD4 or anti-CD8 for 30minutes before the addition of FLAG-HMK-IL-15.

[0014] FIGS. 5A-5C are a series of plots generated by fluorescenceactivated cell sorting (FACS) analysis of PHA-activated PBMCs stainedwith FLAG-HMK-IL-15 proteins and anti-CD25 (IL-2Rá chain) monoclonalantibodies. FIG. 5A demonstrates the existence of IL-2Râ⁺ cells that donot bind FLAG-HMK-IL-15. FIG. 5B demonstrates that almost all PBMCsstimulated with PHA for only one day express either IL-15Rá or IL-2Râsubunits, but not both. FIG. 5C demonstrates that a larger population ofIL-15Rá+ and IL-2Râ+ cells (double positive) are present 3 daysfollowing PHA stimulation.

[0015] FIGS. 6A-6D are a series of plots generated by fluorescenceactivated cell sorting (FACS). To determine the effect ofimmunosuppressant drugs on the mitogen-induced expression of IL-15Rá,PBMCs were preincubated with cyclosporin (CsA; FIG. 6B), rapamycin(PAPA; FIG. 6C), or dexamethasone (Dex; FIG. 6D) for 20 minutes beforethe addition of PHA and then cultured for 3 days. The control,stimulation with PHA only, is shown in FIG. 6A. The level of IL-15Ráexpression was detected using FLAG-HMK-IL-15, anti-FLAG biotinylated Ab,streptavidin-RED670, and FACS analysis.

[0016]FIG. 7 is a bar graph depicting the proliferative response ofhuman PBMCs that were pre-treated with immunosuppressive drugs and PHAand then treated with PHA, IL-2 or IL-15. Cells were harvested after a 4hour pulse of [³H]-TdR and cell-incorporated radioactivity was measuredin a scintillation counter.

[0017]FIG. 8A is a diagram of the protocol used to induce IL-2 and IL-15responsive PBMCs.

[0018]FIG. 8B is a graph depicting the proliferative response of PBMCsthat were pre-stimulated with PHA for 3 days and then cultured in thepresence of IL-2 (Î; ˜) or IL-15 (>; _) for 2 days, and then tested forsecondary response to IL-2 or IL-15 according to the protocolillustrated in FIG. 8A. Arrow indicates that the cytokine was presentduring secondary stimulation. Error bars indicate the mean ″ standarddeviation.

[0019]FIG. 9 is a line graph. PHA-stimulated PBMCs were incubated withgenistein at various doses for 15 minutes at 37EC followed bystimulation with IL-2 or IL-15 for 38 hours in a standard proliferationassay. Control cells were not treated with genistein. Error barsindicate the mean ″ standard deviation.

[0020]FIG. 10 is a graph depicting the effect of rapamycin on theproliferation of PHA-stimulated PBMCs. Cells were incubated withincreasing concentrations of rapamycin for 15 minutes at 37EC, followedby stimulation with IL-2 (_ - - - _) or IL-15 (˜ - - - ˜) for 38 hours,and a standard proliferation assay was performed. In the controlexperiment, cells were cultured in media lacking exogenously addedcytokines. Error bars indicate the mean ″ standard deviation.

[0021]FIG. 11 is a graph depicting the effect of cyclosporin-A on theproliferation of PHA-stimulated PBMCs. The cells were incubated withincreasing concentrations of cyclosporine-A for 15 minutes at 37EC,followed by stimulation with IL-2 or IL-15 for 38 hours, and a standardproliferation assay was performed.

[0022] In the control experiment, cells were cultured in media lackingexogenously added cytokines. Error bars indicate the mean ″ standarddeviation.

[0023]FIGS. 12A and 12B is a pair of graphs depicting the proliferativeresponse of BAF-BO3 cells that express the wild-type IL-2Râ subunit(WT, > - - - >), the mutant IL-2Râ subunit that lacks serine-rich region(S⁻, _ - - - _), or a control vector (V, C - - - C). The cells wereincubated with IL-2 (upper graph) or IL-15 (lower graph) for 38 hoursand a standard proliferation assay was performed. Error bars indicatethe mean ″ standard deviation.

[0024]FIG. 13 is a representation of the wild-type IL-15 nucleic acidand predicted amino acid sequence.

[0025]FIG. 14 is a representation of the mutant IL-15 nucleic acid andpredicted amino acid sequence. The wild-type codon encoding glutamine atposition 149¹, CAG, and the wild-type codon encoding glutamine atposition 156, CAA, have both been changed to GAC, which encodesaspartate. (These positions (149 and 156) correspond to positions 101and 108, respectively, in the mature IL-15 polypeptide, which lacks a48-amino acid signal sequence).

[0026]FIG. 15 is a bar graph depicting the proliferative response ofBAF-BO3 cells cultured in the presence of IL-15 related polypeptides.Abbreviations: WT=BAF-BO3 cells transfected with Prccmv-IL-2Râ (encodingthe human IL-2Râ subunit); V=BAF-BO3 cells transfected with Prccmv-0(plasmid without insert); none=incubation of cells in medium withoutadded interleukin; DM=incubation of cells in medium containing the IL-15double mutant (FLAG-HMK-IL-15-Q149D-Q156D); 149=incubation of cells inmedium containing the IL-15 single mutant (FLAG-HMK-IL-15-Q149D).

[0027]FIG. 16 is a bar graph depicting the proliferative response ofPHA-stimulated human PBMCs cultured in the presence of IL-15 relatedproteins. Abbreviations: WT=BAF-BO3 cells transfected with Prccmv-IL-2Râ(encoding the human IL-2Râ subunit); V=BAF-BO3 cells transfected withPrccmv-0 (plasmid without insert); none=incubation of cells in mediumwithout added interleukin; DM=incubation of cells in medium containingthe IL-15 double mutant (FLAG-HMK-IL-15-Q149D-Q156D); 149=incubation ofcells in medium containing the IL-15 single mutant(FLAG-HMK-IL-15-Q149D); 156=incubation of cells in medium containing theIL-15 single mutant (FLAG-HMK-IL-15-Q156D).

[0028]FIGS. 17A through 17C are a series of plots demonstrating theability of the FLAG-HMK-IL-15 double mutant to bind PHA-activated humanPBMCs. PHA-activated PBMCs were washed and incubated with medium alone(FIG. 17A) or with FLAG-HMK-IL-15 double mutant (FIG. 17B) followed byanti-FLAG biotinylated Ab and streptavidin-RED670. The stained cellswere analyzed by flow cytometry. In (FIGS. 17A-17C), show overlappedcontrol and mutant lines (control=thin line; FLAG-HMK-IL-15-doublemutant=thick line).

[0029]FIGS. 18A and 18B are a series of plots demonstrating the abilityof the wild-type FLAG-HMK-IL-15 polypeptide to bind leukemia cells. Thecells treated were from the leukemic cell lines MOLT-14, YT, HuT-102,and from cell lines currently being established at Beth Israel Hospital(Boston, Mass.), and designated 2A and 2B. The cultured cells werewashed and incubated with either medium alone (tracing alone) or withmedium containing the FLAG-HMK-IL-15 polypeptide (tracing filled in).The cells were then incubated with the biotinylated anti-FLAG antibodyand stained with RED670-conjugated streptavidin. The stained cells wereanalyzed by flow cytometry.

[0030]FIGS. 19A and 19B are graphs depicting the effects of a mutantIL-15/Fcã2a polypeptide on the development of CIA in DBA/1 mice.Administration of the polypeptide delays the onset and decreases theincidence of CIA. Mcie were immunized with intradermal injection of CIIand rechallenged with CII 21 days later. On the day of challenge, micewere randomly divided into two groups, one receiving dailyintraperitoneal injections of control IgG2a (1.5 ig/mouse (?) or mutantIL-15/Fcã2a (1.5 ig/mouse) (?). Mice were examined every day for diseaseactivity, which was quantified as mean clinical score (FIG. 19A) or meannumber of arthritic paws (FIG. 19B).

DETAILED DESCRIPTION

[0031] Mutant IL-15 polypeptides can suppress IL-15 dependent immuneresponses by selectively inhibiting the activity of cells that bindwild-type IL-15. The mutant can also be used to kill the cells to whichit binds.

[0032] Accordingly, the invention features a substantially purepolypeptide that is a mutant of wild-type IL-15, nucleic acid moleculesthat encode this polypeptide, and cells that express the mutantpolypeptide. Methods of treatment, wherein a mutant IL-15 polypeptide isadministered to suppress the immune response (for example, in the eventof an autoimmune disease) or to kill IL-15-binding cells (for example,in the event of unwanted cellular proliferation of IL-15-binding cells)are also within the scope of the invention.

[0033] Mutant IL-15 Polypeptides

[0034] The invention features a substantially pure polypeptide that is amutant of wild-type IL-15, and which can function as an antagonist ofwild-type IL-15. The “wild-type IL-15 polypeptide” referred to herein isa polypeptide that is identical to a naturally-occurring IL-15(interleukin-15) polypeptide. IL-15 has been characterized as a T cellgrowth factor that stimulates the proliferation and differentiation of Bcells, T cells, natural killer (NK) cells, and lymphocyte-activatedkiller (LAK) cells in vitro. In contrast, a “mutant IL-15 polypeptide”is a polypeptide that has at least one mutation relative to wild-typeIL-15 and that inhibits any in vivo or in vitro activity that ischaracteristic of wild-type IL-15.

[0035] A mutant IL-15 polypeptide that can be used according to thepresent invention will generally blocks at least 40%, more preferably atleast 70%, and most preferably at least 90% of one or more of theactivities of the wild-type IL-15 molecule. The ability of a mutantIL-15 polypeptide to block wild-type IL-15 activity can be assessed bynumerous assays, including the BAF-BO3 cell proliferation assaydescribed herein (in which the cells were transfected with a constructencoding IL-2Râ).

[0036] The mutant polypeptides of the invention can be referred to asexhibiting a particular percent identity to wild-type IL-15. Whenexamining the percent identity between two polypeptides, the length ofthe sequences compared will generally be at least 16 amino acids,preferably at least 20 amino acids, more preferably at least 25 aminoacids, and most preferably at least 35 amino acids.

[0037] The term “identity,” as used herein in reference to polypeptideor DNA sequences, refers to the subunit sequence identity between twomolecules. When a subunit position in both of the molecules is occupiedby the same monomeric subunit (i.e., the same amino acid residue ornucleotide), then the molecules are identical at that position. Thesimilarity between two amino acid or two nucleotide sequences is adirect function of the number of identical positions. Thus, apolypeptide that is 50% identical to a reference polypeptide that is 100amino acids long can be a 50 amino acid polypeptide that is completelyidentical to a 50 amino acid long portion of the reference polypeptide.It might also be a 100 amino acid long polypeptide that is 50% identicalto the reference polypeptide over its entire length. Of course, manyother polypeptides will meet the same criteria.

[0038] Sequence identity is typically measured using sequence analysissoftware such as the Sequence Analysis Software Package of the GeneticsComputer Group at the University of Wisconsin Biotechnology Center (1710University Avenue, Madison, Wis. 53705), with the default parametersthereof.

[0039] The mutant polypeptide can be at least 65%, preferably at least80%, more preferably at least 90%, and most preferably at least 95%(e.g., 99%) identical to wild-type IL-15. The mutation can consist of achange in the number or content of amino acid residues. For example, themutant IL-15 can have a greater or a lesser number of amino acidresidues than wild-type IL-15. Alternatively, or in addition, the mutantpolypeptide can contain a substitution of one or more amino acidresidues that are present in the wild-type IL-15. The mutant IL-15polypeptide can differ from wild-type IL-15 by the addition, deletion,or substitution of a single amino acid residue, for example, asubstitution of the residue at position 156. Similarly, the mutantpolypeptide can differ from wild-type by a substitution of two aminoacid residues, for example, the residues at positions 156 and 149. Forexample, the mutant IL-15 polypeptide can differ from wild-type IL-15 bythe substitution of aspartate for glutamine at residues 156 and 149 (asshown in FIGS. 14 and 15). Additions, deletions, and substitutions ofmore than two residues, and substitutions at other positions are likelyto produce a similarly useful (i.e., therapeutically effective) mutantIL-15 polypeptide.

[0040] The substituted amino acid residue(s) can be, but are notnecessarily, conservative substitutions, which typically includesubstitutions within the following groups: glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine.

[0041] The mutations described above can be in the carboxy-terminaldomain of the cytokine, which is believed to bind the IL-2Rã subunit. Itis also possible that one or more mutations can be within the IL-2Râbinding domain.

[0042] In a related aspect, the invention features a chimericpolypeptide that includes a mutant IL-15 polypeptide and a heterologouspolypeptide (i.e., a polypeptide that is not IL-15 or a mutant thereof).The heterologous polypeptide can increase the circulating half-life ofthe chimeric polypeptide in vivo. The polypeptide that increases thecirculating half-life may be a serum albumin, such as human serumalbumin, or the Fc region of the IgG subclass of antibodies that lacksthe IgG heavy chain variable region. The Fc region may include amutation that inhibits complement fixation and Fc receptor binding, orit may be lytic, i.e., able to bind complement or to lyse cells viaanother mechanism, such as antibody-dependent complement lysis (ADCC;08/355,502 filed Dec. 12, 1994).

[0043] The “Fc region” can be a naturally-occurring or syntheticpolypeptide that is homologous to the IgG C-terminal domain produced bydigestion of IgG with papain. IgG Fc has a molecular weight ofapproximately 50 Kda. The polypeptides of the invention can include theentire Fc region, or a smaller portion that retains the ability toextend the circulating half-life of a chimeric polypeptide of which itis a part. In addition, full-length or fragmented Fc regions can bevariants of the wild-type molecule. This is, they can contain mutationsthat may or may not affect the function of the polypeptide; as describedfurther below, native activity is not necessary or desired in all cases.

[0044] The Fc region can be “lytic” or “non-lytic.” A non-lytic Fcregion typically lacks a high affinity Fc receptor binding site and aC′1q binding site. The high affinity Fc receptor binding site of murineIgG Fc includes the Leu residue at position 235 of IgG Fc. Thus, the Fcreceptor binding site can be destroyed by mutating or deleting Leu 235.For example, substitution of Glu for Leu 235 inhibits the ability of theFc region to bind the high affinity Fc receptor. The murine C′1q bindingsite can be functionally destroyed by mutating or deleting the Glu 318,Lys 320, and Lys 322 residues of IgG. For example, substitution of Alaresidues for Glu 318, Lys 320, and Lys 322 renders IgG1 Fc unable todirect antibody-dependent complement lysis. In contrast, a lytic IgG Fcregion has a high affinity Fc receptor binding site and a C′1q bindingsite. The high affinity Fc receptor binding site includes the Leuresidue at position 235 of IgG Fc, and the C′1q binding site includesthe Glu 318, Lys 320, and Lys 322 residues of IgG1. Lytic IgG Fc haswild-type residues or conservative amino acid substitutions at thesesites. Lytic IgG Fc can target cells for antibody dependent cellularcytotoxicity or complement directed cytolysis (CDC). Appropriatemutations for human IgG are also known (see, e.g., Morrison et al., TheImmunologist 2:119-124, 1994; and Brekke et al., The Immunologist 2:125,1994).

[0045] In other instances, the chimeric polypeptide may include themutant IL-15 polypeptide and a polypeptide that functions as anantigenic tag, such as a FLAG sequence. FLAG sequences are recognized bybiotinylated, highly specific, anti-FLAG antibodies, as described herein(see also Blanar et al., Science 256:1014, 1992; LeClair et al., Proc.Natl. Acad. Sci. USA 89:8145, 1992).

[0046] Chimeric polypeptides can be constructed using no more thanconventional molecular biological techniques, which are well within theability of those of ordinary skill in the art to perform.

[0047] As used herein, the terms “protein,” “peptide” and “polypeptide”refer to any chain of amino acid residues, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). The proteins or polypeptides of the invention are“substantially pure, ” meaning that they are at least 60% by weight (dryweight) the polypeptide of interest, for example, a polypeptidecontaining the mutant IL-15 amino acid sequence. Preferably, thepolypeptide is at least 75%, more preferably at least 90%, and mostpreferably at least 99%, by weight, the polypeptide of interest. Puritycan be measured by any appropriate standard method, for example, columnchromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0048] In general, the polypeptides used in the practice of the instantinvention will be synthetic, or produced by expression of a recombinantnucleic acid molecule. Polypeptides that are derived from eukaryoticorganisms or synthesized in E. coli, or other prokaryotes, andpolypeptides that are chemically synthesized will be substantially freefrom some or all of their naturally associated components. In the eventthe polypeptide is a chimera, it can be encoded by a hybrid nucleic acidmolecule (such as those discussed further below) containing one sequencethat encodes all or part of the mutant IL-15, and a second sequence thatencodes all or part of a second gene. For example, the mutant IL-15polypeptide may be fused to a hexa-histidine tag to facilitatepurification of bacterially expressed protein, or to a hemagglutinin tagto facilitate purification of protein expressed in eukaryotic cells.

[0049] The techniques that are required to make mutant IL-15polypeptides are routine in the art, and can be performed without resortto undue experimentation by artisans of ordinary skill. For example, amutation that consists of a substitution of one or more of the aminoacid residues in IL-15 can be created using the PCR-assisted mutagenesistechnique described herein for the creation of mutant IL-15polypeptides. As one non-limiting example, glutamine residues atpositions 149 and 156 can be changed as described below to aspartic acidresidues. Mutations that consists of deletions or additions of aminoacid residues to an IL-15 polypeptide can also be made with standardrecombinant techniques. In the event of a deletion or addition, thenucleic acid molecule encoding IL-15 is simply digested with anappropriate restriction endonuclease. The resulting fragment can eitherbe expressed directly, or manipulated further, for example, by ligatingit to a second fragment. The ligation may be facilitated if the two endsof the nucleic acid molecules contain complementary nucleotides thatoverlap one another, but blunt-ended fragments can also be ligated.

[0050] In addition to generating mutant polypeptides via expression ofnucleic acid molecules that have been altered by recombinant molecularbiological techniques, mutant polypeptides can be chemicallysynthesized. Chemically synthesized polypeptides are routinely generatedby those of skill in the art.

[0051] Procedures for Screening Mutant IL-15 Polypeptides

[0052] Candidate mutants made, for example, as described above can bescreened for the requisite activity. In addition to testing a candidatemutant IL-15 polypeptide in the in vitro assays described in theexamples below, the following procedures can be employed to determinewhether a mutant IL-15 polypeptide is capable of functioning as anantagonist of IL-15 in vivo.

[0053] Transplantation Paradigms

[0054] In order to determine whether a polypeptide is capable offunctioning as an immunosuppressant, it can be administered, eitherdirectly or by genetic therapy, in the context of well-establishedtransplantation paradigms.

[0055] A putative immunosuppressive polypeptide, or a nucleic acidmolecule encoding it, could be systemically or locally administered bystandard means to any conventional laboratory animal, such as a rat,mouse, rabbit, guinea pig, or dog, before an allogeneic or xenogeneicskin graft, organ transplant, or cell implantation is performed on theanimal. Strains of mice such as C57B1-10, B10.BR, and B10.AKM (JacksonLaboratory, Bar Harbor, Me.), which have the same genetic background butare mismatched for the H-2 locus, are well suited for assessing variousorgan grafts.

[0056] Heart Transplantation

[0057] A method for performing cardiac grafts by anastomosis of thedonor heart to the great vessels in the abdomen of the host was firstpublished by Ono et al. (J. Thorac. Cardiovasc. Surg. 57:225, 1969; seealso Corry et al., Transplantation 16:343, 1973). According to thissurgical procedure, the aorta of a donor heart is anastomosed to theabdominal aorta of the host, and the pulmonary artery of the donor heartis anastomosed to the adjacent vena cava using standard microvasculartechniques. Once the heart is grafted in place and warmed to 37EC withRinger's lactate solution, normal sinus rhythm will resume. Function ofthe transplanted heart can be assessed frequently by palpation ofventricular contractions through the abdominal wall. Rejection isdefined as the cessation of myocardial contractions, which can beconfirmed by under anesthesia. Mutant IL-15 polypeptides would beconsidered effective in reducing organ rejection if hosts that receivedinjections, for example, of the mutant IL-15 polypeptide experiencedlonger heart engraftment than the untreated hosts.

[0058] Skin Grafting

[0059] The effectiveness of mutant IL-15 polypeptides can also beassessed following a skin graft. To perform skin grafts on a rodent, adonor animal is anesthetized and the full thickness skin is removed froma part of the tail. The recipient animal is also anesthetized, and agraft bed is prepared by removing a patch of skin from the shaved flank.Generally, the patch is approximately 0.5×0.5 cm. The skin from thedonor is shaped to fit the graft bed, positioned, covered with gauze,and bandaged. The grafts can be inspected daily beginning on the sixthpost-operative day, and are considered rejected when more than half ofthe transplanted epithelium appears to be non-viable. Mutant IL-15polypeptides would be considered effective in reducing rejection of theskin grafts if hosts that received injections, for example, of themutant IL-15 polypeptide tolerated the graft longer than the untreatedhosts.

[0060] Islet Allograft Model

[0061] DBA/2J islet cell allografts can be transplanted into rodents,such as 6-8 week-old IL-4^(−/−) and IL-4^(+/−) mice rendered diabetic bya single intraperitoneal injection of streptozotocin (225 mg/kg; SigmaChemical Co., St. Louis, Mo.). As a control, syngeneic IL-4^(−/−) isletcell grafts can be transplanted into diabetic IL-4^(−/−) mice. Isletcell transplantation can be performed by following published protocols(e.g., see Gotoh et al., Transplantation 42:387, 1986). Briefly, donorpancreata are perfused in situ with type IV collagenase (2 mg/ml;Worthington Biochemical Corp., Freehold, N.J.). After a 40 minutedigestion period at 37EC, the islets are isolated on a discontinuousFicoll gradient. Subsequently, 300-400 islets are transplanted under therenal capsule of each recipient. Allograft function can be followed byserial blood glucose measurements (Accu-Check III?; Boehringer,Mannheim, Germany). Primary graft function is defined as a blood glucoselevel under 11.1 mmol/l on day 3 post-transplantation, and graftrejection is defined as a rise in blood glucose exceeding 16.5 mmol/l(on each of at least 2 successive days) following a period of primarygraft function.

[0062] Models of Autoimmune Disease

[0063] Models of autoimmune disease provide another means to assesspolypeptides in vivo. These models are well known to skilled artisansand can be used to determine whether a given mutant IL-15 polypeptide isan immunosuppressant that would be therapeutically useful in treating aspecific autoimmune disease when delivered either directly or viagenetic therapy.

[0064] Autoimmune diseases that have been modeled in animals includerheumatic diseases, such as rheumatoid arthritis (see, e.g., the Exampleheaded “Induction of Collagen-induced Arthritis” below) and systemiclupus erythematosus (SLE), type I diabetes, and autoimmune diseases ofthe thyroid, gut, and central nervous system. For example, animal modelsof SLE include MRL mice, BXSB mice, and NZB mice and their F₁ hybrids.These animals can be crossed in order to study particular aspects of therheumatic disease process; progeny of the NZB strain develop severelupus glomerulonephritis when crossed with NZW mice (Bielschowsky etal., Proc. Univ. Otago Med. Sch. 37:9, 1959; see also FundamentalImmunology, Paul, Ed., Raven Press, New York, N.Y., 1989). Similarly, ashift to lethal nephritis is seen in the progeny of NBZ×SWR matings(Data et al., Nature 263:412, 1976). The histological appearance ofrenal lesions in SNF₁ mice has been well characterized (Eastcott et al.,J. Immunol. 131:2232, 1983; see also Fundamental Immunology, supra).Therefore, the general health of the animal as well as the histologicalappearance of renal tissue can be used to determine whether theadministration of a mutant IL-15 polypeptide can effectively suppressthe immune response in an animal model of SLE.

[0065] One of the MRL strains of mice that develops SLE, MRL-1pr/1pr,also develops a form of arthritis that resembles rheumatoid arthritis inhumans (Theofilopoulos et al., Adv. Immunol. 37:269, 1985).Alternatively, an experimental arthritis can be induced in rodents byinjecting rat type II collagen (2 mg/ml) mixed 1:1 in Freund's completeadjuvant (100 il total) into the base of the tail. Arthritis develops2-3 weeks after immunization. The ability of nucleic acid moleculesencoding mutant IL-15 polypeptides to combat the arthritic condition canbe assessed by targeting the nucleic acid molecules to T lymphocytes.One way to target T lymphocytes is the following: spleen cellsuspensions are prepared 2-3 days after the onset of arthritis andincubated with collagen (100 ig/ml) for 48 hours to induce proliferationof collagen-activated T cells. During this time, the cells aretransduced with a vector encoding the polypeptide of interest. As acontrol, parallel cultures are grown but not transduced, or aretransduced with the “empty” vector. The cells are then injectedintraperiotoneally (5×10⁷ cells/animal). The effectiveness of thetreatment is assessed by following the disease symptoms during thesubsequent 2 weeks, as described by Chernajovsky et al. (Gene Therapy2:731-735, 1995). Lesser symptoms, compared to control, indicate thatthe peptide of interest, and the nucleic acid molecule encoding it,function as an immunosuppressant potentially useful in the treatment ofautoimmune disease.

[0066] The ability of a mutant IL-15 polypeptide to suppress the immuneresponse in the case of Type I diabetes can be tested in the BB ratstrain, which was developed from a commercial colony of Wistar rats atthe Bio-Breeding Laboratories in Ottawa. These rats spontaneouslydevelop autoantibodies against islet cells and insulin, just as occurswith human Type I diabetes. Alternatively, NOD (non-obese diabetic) micecan be used as a model system.

[0067] Autoimmune diseases of the thyroid have been modeled in thechicken. Obese strain (OS) chickens consistently develop spontaneousautoimmune thyroiditis resembling Hashimoto's disease (Cole et al.,Science 160:1357, 1968). Approximately 15% of these birds produceautoantibodies to parietal cells of the stomach, just as in the humancounterpart of autoimmune thyroiditis. The manifestations of the diseasein OS chickens, which could be monitored in the course of mutant IL-15polypeptide treatment, include body size, fat deposit, serum lipids,cold sensitivity, and infertility.

[0068] Models of autoimmune disease in the central nervous system (CNS)can be experimentally induced. An inflammation of the CNS, which leadsto paralysis, can be induced by a single injection of brain or spinalcord tissue with adjuvant in many different laboratory animals,including rodents and primates. This model, referred to as experimentalallergic encephalomyelitis (EAE), is T cell mediated. Similarly,experimentally induced myasthenia gravis can be produced by a singleinjection of acetylcholine receptor with adjuvants (Lennon et al., Ann.N.Y. Acad. Sci. 274:283, 1976).

[0069] Autoimmune diseases of the gut can be modeled in IL-2 or IL-10“knock out” mice, or in mice that receive enemas containing bovine serumalbumin.

[0070] Nucleic Acid Molecules Encoding Mutant IL-15

[0071] The mutant IL-15 polypeptide, either alone or as a part of achimeric polypeptide, such as those described above, can be obtained byexpression of a nucleic acid molecule. Thus, nucleic acid moleculesencoding polypeptides containing a mutant IL-15 are considered withinthe scope of the invention. Just as mutant IL-15 polypeptides can bedescribed in terms of their identity with wild-type IL-15 polypeptides,the nucleic acid molecules encoding them will necessarily have a certainidentity with those that encode wild-type IL-15. For example, thenucleic acid molecule encoding a mutant IL-15 polypeptide can be atleast 65%, preferably at least 75%, more preferably at least 85%, andmost preferably at least 95% (e.g., 99%) identical to the nucleic acidencoding wild-type IL-15. For nucleic acids, the length of the sequencescompared will generally be at least 50 nucleotides, preferably at least60 nucleotides, more preferably at least 75 nucleotides, and mostpreferably 110 nucleotides.

[0072] The nucleic acid molecules of the invention can contain naturallyoccurring sequences, or sequences that differ from those that occurnaturally, but, due to the degeneracy of the genetic code, encode thesame polypeptide. These nucleic acid molecules can consist of RNA or DNA(for example, genomic DNA, cDNA, or synthetic DNA, such as that producedby phosphoramidite-based synthesis), or combinations or modifications ofthe nucleotides within these types of nucleic acids. In addition, thenucleic acid molecules can be double-stranded or single-stranded (i.e.,either a sense or an antisense strand).

[0073] The nucleic acid molecules of the invention are referred to as“isolated” because they are separated from either the 5′ or the 3′coding sequence with which they are immediately contiguous in thenaturally occurring genome of an organism. Thus, the nucleic acidmolecules are not limited to sequences that encode polypeptides; some orall of the non-coding sequences that lie upstream or downstream from acoding sequence can also be included. Those of ordinary skill in the artof molecular biology are familiar with routine procedures for isolatingnucleic acid molecules. They can, for example, be generated by treatmentof genomic DNA with restriction endonucleases, or by performance of thepolymerase chain reaction (PCR). In the event the nucleic acid moleculeis a ribonucleic acid (RNA), molecules can be produced by in vitrotranscription.

[0074] The isolated nucleic acid molecules of the invention can includefragments not found as such in the natural state. Thus, the inventionencompasses recombinant molecules, such as those in which a nucleic acidsequence (for example, a sequence encoding a mutant IL-15) isincorporated into a vector (e.g., a plasmid or viral vector) or into thegenome of a heterologous cell (or the genome of a homologous cell, at aposition other than the natural chromosomal location).

[0075] As described above, the mutant IL-15 polypeptide of the inventionmay exist as a part of a chimeric polypeptide. In addition to, or inplace of, the heterologous polypeptides described above, a nucleic acidmolecule of the invention can contain sequences encoding a “marker” or“reporter.” Examples of marker or reporter genes include â-lactamase,chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo^(r), G418^(r)), dihydrofolatereductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidinekinase (TK), lacZ (encoding â-galactosidase), and xanthine guaninephosphoribosyltransferase (XGPRT). As with many of the standardprocedures associated with the practice of the invention, skilledartisans will be aware of additional useful reagents, for example, ofadditional sequences that can serve the function of a marker orreporter.

[0076] The nucleic acid molecules of the invention can be obtained byintroducing a mutation into IL-15-encoding DNA obtained from anybiological cell, such as the cell of a mammal. Thus, the nucleic acidsof the invention can be those of a mouse, rat, guinea pig, cow, sheep,horse, pig, rabbit, monkey, baboon, dog, or cat. Preferably, the nucleicacid molecules will be those of a human.

[0077] Expression of Mutant IL-15 Gene Products

[0078] The nucleic acid molecules described above can be containedwithin a vector that is capable of directing their expression in, forexample, a cell that has been transduced with the vector. Accordingly,in addition to mutant IL-15 polypeptides, expression vectors containinga nucleic acid molecule encoding a mutant IL-15 polypeptide and cellstransfected with these vectors are among the preferred embodiments.

[0079] Vectors suitable for use in the present invention includeT7-based vectors for use in bacteria (see, for example, Rosenberg etal., Gene 56:125, 1987), the pMSXND expression vector for use inmammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), andbaculovirus-derived vectors (for example the expression vector pBacPAK9from Clontech, Palo Alto, Calif.) for use in insect cells. The nucleicacid inserts, which encode the polypeptide of interest in such vectors,can be operably linked to a promoter, which is selected based on, forexample, the cell type in which expression is sought. For example, a T7promoter can be used in bacteria, a polyhedrin promoter can be used ininsect cells, and a cytomegalovirus or metallothionein promoter can beused in mammalian cells. Also, in the case of higher eukaryotes,tissue-specific and cell type-specific promoters are widely available.These promoters are so named for their ability to direct expression of anucleic acid molecule in a given tissue or cell type within the body.Skilled artisans are well aware of numerous promoters and otherregulatory elements which can be used to direct expression of nucleicacids.

[0080] In addition to sequences that facilitate transcription of theinserted nucleic acid molecule, vectors can contain origins ofreplication, and other genes that encode a selectable marker. Forexample, the neomycin-resistance (neo^(r)) gene imparts G418 resistanceto cells in which it is expressed, and thus permits phenotypic selectionof the transfected cells. Those of skill in the art can readilydetermine whether a given regulatory element or selectable marker issuitable for use in a particular experimental context.

[0081] Viral vectors that can be used in the invention include, forexample, retroviral, adenoviral, and adeno-associated vectors, herpesvirus, simian virus 40 (SV40), and bovine papilloma virus vectors (see,for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH LaboratoryPress, Cold Spring Harbor, New York).

[0082] Prokaryotic or eukaryotic cells that contain and express anucleic acid molecule that encodes a mutant IL-15 polypeptide are alsofeatures of the invention. A cell of the invention is a transfectedcell, i.e., a cell into which a nucleic acid molecule, for example anucleic acid molecule encoding a mutant IL-15 polypeptide, has beenintroduced by means of recombinant DNA techniques. The progeny of such acell are also considered within the scope of the invention. The precisecomponents of the expression system are not critical. For example, amutant IL-15 polypeptide can be produced in a prokaryotic host, such asthe bacterium E. coli, or in a eukaryotic host, such as an insect cell(e.g., an Sf21 cell), or mammalian cells (e.g., COS cells, NIH 3T3cells, or HeLa cells). These cells are available from many sources,including the American Type Culture Collection (Manassas, Va.). Inselecting an expression system, it matters only that the components arecompatible with one another. Artisans or ordinary skill are able to makesuch a determination. Furthermore, if guidance is required in selectingan expression system, skilled artisans may consult Ausubel et al.(Current Protocols in Molecular Biology, John Wiley and Sons, New York,N.Y., 1993) and Pouwels et al. (Cloning Vectors: A Laboratory Manual,1985 Suppl. 1987).

[0083] The expressed polypeptides can be purified from the expressionsystem using routine biochemical procedures, and can be used asdiagnostic tools or as therapeutic agents, as described below.

[0084] Mutant IL-15 Polypeptides in Diagnosis

[0085] The polypeptide of the invention can be used to diagnose apatient as having a disease amenable to treatment with an IL-15antagonist. According to this method, a sample of tissue is obtainedfrom the patient and exposed to an antigenically-tagged mutant IL-15polypeptide. The sample may be any biological sample, such as a blood,urine, serum, or plasma sample. In addition, the sample may be a tissuesample (e.g., biopsy tissue), or an effusion obtained from a joint(e.g., synovial fluid), from the abdominal cavity (e.g., ascites fluid),from the chest (e.g., pleural fluid), from the central nervous system(e.g., cerebral spinal fluid), or from the eye (e.g., aqueous humor).The sample may also consist of cultured cells that were originallyobtained from a patient (e.g., peripheral blood mononuclear cells). Itis expected that the sample will be obtained from a mammal, andpreferably, that the mammal will be a human patient. If the samplecontains cells that are bound by the polypeptide described (i.e., anantigenically-tagged mutant IL-15 polypeptide), it is highly likely thatthey would be bound by a mutant IL-15 polypeptide in vivo and therebyinhibited from proliferating, or even destroyed, in vivo. The presentingsymptoms of candidate patients for such testing and the relevant tissuesto be sampled given a particular set of symptoms are known to thoseskilled in the field of immunology.

[0086] Modulation of the Immune Response

[0087] Also featured in the invention is a method of suppressing theimmune response in a patient by administering a dose of mutant IL-15that is sufficient to competitively bind the IL-15 receptor complex, andthereby modulate IL-15 dependent immune responses. The polypeptideadministered can be a mutant IL-15 polypeptide, including those that area part of the chimeric polypeptides described above. This method may beused to treat a patient who is suffering from an autoimmune disease,including but not limited to the following: (1) a rheumatic disease suchas rheumatoid arthritis, systemic lupus erythematosus, Sjögren'ssyndrome, scleroderma, mixed connective tissue disease, dermatomyositis,polymyositis, Reiter's syndrome or Behcet's disease (2) type II diabetes(3) an autoimmune disease of the thyroid, such as Hashimoto'sthyroiditis or Graves' Disease (4) an autoimmune disease of the centralnervous system, such as multiple sclerosis, myasthenia gravis, orencephalomyelitis (5) a variety of phemphigus, such as phemphigusvulgaris, phemphigus vegetans, phemphigus foliaceus, Senear-Ushersyndrome, or Brazilian phemphigus, (6) psoriasis, and (7) inflammatorybowel disease (e.g., ulcerative colitis or Crohn's Disease). Theadministration of the mutant IL-15 polypeptide of the invention may alsobe useful in the treatment of acquired immune deficiency syndrome(AIDS). Similarly, the method may be used to treat a patient who hasreceived a transplant of biological materials, such as an organ, tissue,or cell transplant. In addition, patients who have received a vascularinjury would benefit from this method.

[0088] Induction of a Lytic Response

[0089] Through the administration of a lytic form of a mutant IL-15chimeric polypeptide, it is possible to selectively kill autoreactive or“transplant destructive” immune cells without massive destruction ofnormal T cells. Accordingly, the invention features a method of killingautoreactive or “transplant destructive” immune cells or any malignantcell that expresses the IL-15 receptor in vivo. The method is carriedout by administering to a patient an amount of mutant IL-15 linked to apolypeptide that is sufficient to activate the complement system, lysecells by the ADCC mechanism, or otherwise kill cells expressing thewild-type IL-15 receptor complex. This method can be used, for example,to treat patients who have IL-15R⁺ leukemia, lymphoma, or other IL-15R⁺malignant diseases, such as colon cancer.

[0090] Formulations for Use and Routes of Administration

[0091] In therapeutic applications, the polypeptide may be administeredwith a physiologically-acceptable carrier, such as physiological saline.The therapeutic compositions of the invention can also contain a carrieror excipient, many of which are known to skilled artisans. Excipientsthat can be used include buffers (e.g., citrate buffer, phosphatebuffer, acetate buffer, and bicarbonate buffer), amino acids, urea,alcohols, ascorbic acid, phospholipids, proteins (e.g., serum albumin),EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. Thepolypeptides of the invention can be formulated in various ways,according to the corresponding route of administration. For example,liquid solutions can be made for ingestion or injection; gels or powderscan be made for ingestion, inhalation, or topical application. Methodsfor making such formulations are well known and can be found in, forexample, “Remington's Pharmaceutical Sciences.

[0092] Routes of administration are also well known to skilledpharmacologists and physicians and include intraperitoneal,intramuscular, subcutaneous, and intravenous administration. Additionalroutes include intracranial (e.g., intracisternal or intraventricular),intraorbital, opthalmic, intracapsular, intraspinal, intraperitoneal,transmucosal, topical, subcutaneous, and oral administration. It isexpected that the intravenous route will be preferred for theadministration of mutant IL-15 polypeptides. The subcutaneous route mayalso be used frequently as the subcutaneous tissue provides a stableenvironment for the polypeptide, from which it can be slowly released.Another route of administration of mutant IL-15 polypeptides that hasproven feasible is the intraperitoneal route. As described in theexamples below, intraperitoneal injection of mutant IL-15/Fcpolypeptides is an effective treatment for collagen-induced arthritis(i.e., it delays the onset and decreases the incidence and severity ofcollagen-induced arthritis). These results demonstrate that systemicadministration of mutant IL-15 polypeptides is effective. Thus, whileone may target the peptides more specifically to their site of action,such targeting is not necessary for effective treatment. For example, inthe event of an autoimmune disease such as rheumatoid arthritis, whichaffects the joints, mutant IL-15 polypeptides need not be targetedspecifically to the joints.

[0093] The polypeptides of the invention are less likely to beimmunogenic than many therapeutic polypeptides because they can differfrom a wild-type polypeptide by only a few amino acid residues. Inaddition, the mutant IL-15 polypeptides are targeted to cells thatexpress abundant receptors to which they can bind with high affinity.

[0094] It is well known in the medical arts that dosages for any onepatient depend on many factors, including the general health, sex,weight, body surface area, and age of the patient, as well as theparticular compound to be administered, the time and route ofadministration, and other drugs being administered concurrently. Dosagesfor the polypeptide of the invention will vary, but can, whenadministered intravenously, be given in doses of approximately 0.01 mgto 100 mg/ml blood volume. A dosage can be administered one or moretimes per day, if necessary, and treatment can be continued forprolonged periods of time, up to and including the lifetime of thepatient being treated. If a polypeptide of the invention is administeredsubcutaneously, the dosage can be reduced, and/or the polypeptide can beadministered less frequently. Determination of correct dosage for agiven application is well within the abilities of one of ordinary skillin the art of pharmacology. In addition, those of ordinary skill in theart can turn to the Examples presented below for guidance in developingan effective treatment regime. For example, one could begin tailoringthe dosage of mutant IL-15 polypeptides required for effective treatmentof humans from the dosage proven effective in the treatment of smallmammals. Mice were effectively treated for an autoimmune condition withdaily intraperitoneal injections containing 1.5 ig of mutantIL-15/Fcã2a. Of course, only routine experimentation would be requiredto more precisely define the effective limits of any givenadministrative regime. For example, in a conservative approach, onecould define the lowest effective dosage in small mammals, andadminister that dose to progressively larger mammals before beginninghuman safety trials.

EXAMPLES

[0095] Reagents

[0096] The following reagents were used in the studies described herein:recombinant human IL-2 was obtained from Hoffman-La Roche (Nutley,N.J.); rapamycin was obtained from Wyeth-Ayerst (Princeton, N.J.);cyclosporine-A (CsA) was obtained from Sandoz (East Hanover, N.J.);RPMI-1640 and fetal calf serum (FCS) were obtained from BioWittaker(Walkersville, Md.); penicillin, streptomycin, G418, andstrepavidin-RED670 were obtained from Gibco-BRL (Gaithersburg, Md.);dexamethasone, PHA, lysozyme, Nonidet P-40, NaCl, HEPES, and PMSF wereobtained from Sigma (St. Louis, Mo.); Ficoll-Hypaque was obtained fromPharmacia Biotech (Uppsala, Sweden); recombinant human IL-15 andanti-human IL-15 Ab were obtained from PeproTech (Rocky Hill, N.J.);anti-FLAG Ab and anti-FLAG-affinity beads were obtained fromInternational Biotechnologies, Inc. (Kodak, New Haven, Conn.); pRcCMVwas obtained from InVitrogen Corporation (San Diego, Calif.); genisteinwas obtained from ICN Biomedicals (Irvine, Calif.); disuccinimidylsuberate (DSS) was obtained from Pierce (Rockford, Ill.); restrictionendonucleases were obtained from New England Biolabs (Beverly, Mass.);[³H]TdR was obtained from New England Nuclear (Boston, Mass.); andfluorescent dye conjugated antibodies CD25-PE³, CD14-PE, CD16-PE,CD122-PE, CD4-FITC, CD8-FITC, IgG1-PE or IgG1-FITC were obtained fromBeckton/Dickinson (San Jose, Calif.). FLAG peptide was synthesized inthe Peptide Synthesis Facility at Harvard Medical School.

[0097] Production of FLAG-HMK-IL-15 Fusion Protein

[0098] To study the cellular pattern of human IL-15 receptor expression,a plasmid that could be used to express an IL-15 fusion protein wasconstructed. The plasmid encodes an IL-15 polypeptide having anN-terminus covalently bound to the 18 amino acid FLAG-HMK-sequence(FLAG-HMK-IL-15). FLAG sequences are recognized by biotinylated, highlyspecific anti-FLAG antibodies (Blanar et al., Science 256:1014, 1992);LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145,1992) while HMK(Heart Muscle Kinase recognition site) sequences allow introduction ofradioactive label [³²P] into the molecule (Blanar et al., supra, LeClairet al., supra).

[0099] For the construction of the plasmid FLAG-HMK-IL-15, a 322 bp cDNAfragment encoding mature IL-15 protein was amplified by PCR utilizingsynthetic oligonucleotides [sense 5′-GGAATTCAACTGGGTGAATGTAATA-3′ (SEQID NO: 1; EcoRI site (underlined) plus bases 145-162); antisense5′-CGGGATCCTCAAGAAGTGTTGATGAA-3′ (SEQ ID NO: 2; BamHI site [underlined]plus bases 472-489)]. The template DNA was obtained from PHA-activatedhuman PBMCs. The PCR product was purified, digested with EcoRI andBamHI, and cloned into the pAR(DRI) 59/60 plasmid digested withEcoRI-BamHI as described (Blanar et al., Science 256:1014, 1992; LeClairet al., Proc. Natl. Acad. Sci. USA 89:8145, 1992). The backbone of thepAR(DRI) 59/60 plasmid contains in frame sequences encoding the FLAG andHMK recognition peptide sequences (Blanar et al., Science 256:1014,1992; LeClair et al., Proc. Natl. Acad. Sci. USA 89:8145, 1992).

[0100] Expression and Purification of FLAG-HMK-IL-15 Fusion Protein

[0101] The IL-15-related fusion construct, FLAG-HMK-IL-15, was expressedin BL-21 strain E. coli and affinity purified with anti-FLAG coatedbeads as described (Blanar et al., Science 256:1014, 1992; LeClair etal., Proc. Natl. Acad. Sci. USA 89:8145, 1992). The fusion protein waseluted from affinity columns after extensive washing with 0.1 M glycine(pH 3.0). The eluate containing FLAG-HMK-IL-15 was dialyzed against abuffer containing 50 mM Tris (pH 7.4) and 0.1 M NaCl for 18 hours at4EC, filtered through a 0.2 im membrane, and stored at−20EC.

[0102] Analysis of FLAG-HMK-IL-15 Fusion Protein

[0103] The authenticity of FLAG-HMK-IL-15 fusion protein was confirmedas follows. The proteins present in eluates from the affinity columnwere separated by SDS-PAGE, transferred to PVDF membranes, and stainedwith Coomassie blue (FIG. 1, lane a). The presence of FLAG sequences wasspecifically examined using anti-FLAG Ab (FIG. 1, lane b), and thepresence of hIL-15 sequences was specifically examined using anti-hIL-15Ab (FIG. 1, lane c). A 15 kDa protein, whose mass corresponds to theexpected size of FLAG-HMK-IL-15 protein, was identified in each of theseanalyses. To confirm that the fusion protein contains the HMKrecognition site, eluates from the affinity column were incubated in thepresence of enzymatically active HMK and radioactive [³²P]-ã-ATP,followed by separation of proteins by SDS-PAGE. Autoradiography againidentified a single radiolabeled 15 kDa band that co-migrates with theFLAG-HMK-IL-15 (FIG. 1, lane d). Thus, the 15 kDa protein contains allthree elements of the designed fusion protein: FLAG sequence, HMKsequence, and hIL-15 sequence.

[0104] Cell Culture

[0105] Human PBMCs

[0106] Human peripheral blood mononuclear cells (PBMCs) were obtainedfrom leukophoresis preparations from healthy donors and isolated byFicoll-Hypaque density gradient centrifugation according to standardprotocols.

[0107] PBMCs were maintained at 37EC in an atmosphere containing 5% CO₂in RPMI-1640 medium supplemented with 10% FCS, penicillin (0.5 U/ml),and streptomycin (0.5 ig/ml).

[0108] For studies involving rapamycin or genistein, human PBMCs(4×10⁵/well) were plated in U-bottomed 96-well tissue culture plates(NUNC; Naperville, Ill.) with rapamycin (10 ng/ml), genistein (10 ig/ml)or media alone, and cultured for 3 days at 37EC and 5% CO₂. Cellviability was tested utilizing the standard trypan blue exclusion method(Current Protocols in Immunology, Vol. 3, ed. R. Coico, New York: JohnWiley & Sons, 1995).

[0109] BAF-BO3 Cells

[0110] The BAF-BO3 cell line is an IL-3-dependent, murine pro-B cellline. A BAF-BO3 cell line that was pre-selected for high expression ofthe IL-2Rá subunit was kindly provided by T. Taniguchi (Osaka, Japan;Hatakeyama et al., Cell 59:837, 1989). BAF-BO3 cells were transfectedwith vector control (pRcCMV-0) or cDNA encoding the human IL-2Râ subunitcloned into pRcCMV (pRcCMV-IL-2Râ), by electroporation at 350 volts and500 iFarads using 20 ig of ScaI linearized plasmid. Transfected cellswere selected in the presence of 1 mg/ml G418 and tested for theexpression of the IL-2Râ subunit by fluorescence activated cell sorting(FACS) analysis as described (Maslinski et al., Int. Immunol. 4:509,1992; Hataleyama et al., Cell 59:837, 1989).

[0111] BAF-BO3 cells were maintained at 37EC with 5% CO₂ in RPMI-1640medium supplemented with 10% FCS, penicillin (0.5 U/ml), streptomycin(0.5 ig/ml), and 5% (V/V) IL-3 rich WEHI cell supernatant (Hatakeyama etal., Cell 59:837, 1989).

[0112] To test mutant polypeptides, a cytotoxic T lymphocyte cell line,CTLL-2, can also be used. This cell line is available from the AmericanType Culture Collection (12301 Parklawn Drive, Rockville, Md.20852-1776).

[0113] Cellular Proliferation Assays

[0114] Human PBMCs

[0115] Peripheral blood mononuclear cells (1×10⁶/ml) were stimulatedwith PHA (2 ig/ml) for 72 hours at 37EC in RPMI-1640 medium supplementedwith 10% FCS, penicillin, and streptomycin, as described above. Toinvestigate the effect of immunosuppressive drugs, aliquots of cells(10⁶ PBMCs/ml) were preincubated with cyclosporin-A (CsA; 150 ng/ml),rapamycin (5 ng/ml), or dexamethasone (0.1 mg/ml) for 20 minutes beforethe addition of PHA.

[0116] In some experiments, cells prepared as described above werewashed after 72 hours in culture, plated in U-bottomed 96-well tissueculture plates at 2×10⁵ PBMCs per well (NUNC, Naperville, Ill.), andthen re-stimulated with one of the following reagents: PHA (2 ig/ml),FLAG peptide (10⁻⁴ M), IL-2 (100 U/ml), or FLAG-HMK-IL-15 (100 il/ml)for 72 hours. Cells were “pulsed” for 4 hours with 1 iCi of [³H]TdR (NewEngland Nuclear, Boston, Mass.), harvested onto Whatman 934-AH glassmicrofiber filters, and hypotonically lysed using a PHD Cell Harvester(Cambridge Technology, Inc., Cambridge, Mass.). Cell associated [³H]-TdRwas measured using a Beckman LS 2800 scintillation counter (Beckman,Fullerton, Calif.).

[0117] BAF-BO3 Cells

[0118] IL-3 dependent BAF-BO3 cells, which constitutively express theIL-2Rã subunit, were transfected with a control pRcCMV construct(pRcCMV-0) or a pRcCMV construct containing cDNA encoding thefull-length human IL-2Râ subunit and the neo^(r) gene (pRcCMV-IL-2Râ).G418 resistant clones were then selected. Cells that were selected inthis way were used to test the biological activity of FLAG-HMK-IL-15.This strategy has been used to identify cells transfected with cDNAencoding full-length IL-2Râ subunits, and which, therefore, proliferatein response to exogenously added human IL-2 or IL-15 (Giri et al., EMBOJ. 13:2822, 1994). Cells were washed twice to remove the mediumcontaining the growth factor IL-3, and starved for 16 hours in RPMI-1640medium supplemented with 10% FCS, penicillin (0.5 U/ml), andstreptomycin (0.5 ig/ml). Cells were then plated at 2×10⁴ cells/well,stimulated with FLAG-HMK-IL-15, and cultured for 72 hours at 37EC in anatmosphere containing 5% CO₂. This was followed by a 14 hour “pulse”with 1 iCi of [³H]-TdR. The BAF-BO3 cells were then harvested andcell-associated radioactivity was measured by scintillation counting asdescribed above.

[0119] Cross-linking Activated PBMCs Cultured with FLAG-HMK-IL-15

[0120] After 72 hours in culture, PHA-activated PBMCs were washed twicein cold phosphate-buffered saline (PBS), and 10⁷ cells/ml were incubatedfor 30 minutes at 4EC in 400 il of RMPI-1640 medium supplemented with 25mM HEPES (pH 7.4), 10% FCS, penicillin (0.5 U/ml), and streptomycin (0.5ig/ml). Supplemented RPMI-1640 medium (20 il) containing 1 ig ofrecombinant human IL-15 as a control for non-specific binding or mediumalone was then added, and the incubation was continued for 15 minutesbefore 20 il of medium containing [³²P]-labeled FLAG-HMK-IL-15 (1 ng,500,000 cpm) was added to each sample. After further incubation for 1hour at 4EC, the cells were washed with PBS and resuspended in 250 il ofPBS. Disuccinimidyl suberate (DDS; 1 mM), a cross-linker, was added, andcross-linking was performed as described by Tsudo et al. (Proc. Natl.Acad. Sci. USA 83:9694, 1986). The cross-linked cells were collected bycentrifugation, washed, solubilized with extraction buffer (2% NonidetP-40, 0.14 M NaCl, 25 mM HEPES (pH 7.4), 1 mM phenyl-methylsulfonylfluoride) and centrifuged. Proteins present in the supernatants of thesecell lysates were separated by 8% SDS-PAGE under reducing conditions. Anautoradiogram was developed from the dry gel after exposure at−70ECovernight.

[0121] Cell Staining for Flow Cytometry

[0122] Resting or PHA-activated PBMCs (3×10⁵ cells/tube) were washedtwice with ice-cold PBS/0.02% sodium azide and incubated in mediumcontaining FLAG-HMK-IL-15 (0.1 ig/100 il) or, as a control, in mediumalone. The incubation was carried out on ice for 30 minutes. The cellswere then washed with PBS and incubated for two sequential 30 minuteperiods at 4EC with biotin-conjugated anti-FLAG monoclonal antibody (0.5ig/100 il) and streptavidin-RED670 (0.5 ig/100 il). The cells were thencounterstained with CD25-PE, CD14-PE, CD16-PE, CD122-PE, CD4-FITC, orCD8-FITC, as indicated. Addition of IgG1-PE or IgG1-FITC was added tocell populations that had not been incubated with any of theaforementioned reagents in order to identify non-specific binding. Cellsurface phenotype was analyzed using FACScan (Becton/Dickinson, Calif.)and Cell Quest software.

[0123] FLAG-HMK-IL-15 Binds the IL-15Rá Subunit

[0124] The purified FLAG-HMK-IL-15 fusion protein was tested todetermine whether it interacts with cell surface IL-15 receptors. Asdescribed above, [³²P]-FLAG-HMK-IL-15 was added to cultures of PBMCsthat were activated by a mitogen, PHA. In order to permanently bindinteractive proteins to one another, the chemical cross-linkerdisuccinimidyl suberate (DSS) was added. The cells were washed, lysed,centrifuged, and detergent-soluble proteins were separated by SDS-PAGE.Autoradiography of SDS-PAGE separated proteins revealed a single 75-80kDa band corresponding to the combined molecular weight ofFLAG-HMK-IL-15 (15 kDa) and the human IL-15Rá subunit (60-65 kDa; FIG.2, lane 1). The identity of this band as the IL-15Rá subunit wasconfirmed by cross-linking experiments conducted in the presence of amolar excess of hIL-15. Under these conditions, we failed to detect theradiolabeled 15 kDa band (FIG. 2, lane 2). Thus, the conformation of[³²P]-FLAG-HMK-IL-15 fusion proteins allows site specific binding to the60-65 kDa IL-15Rá subunit expressed on the surface of mitogen-activatedPBMCs.

[0125] FLAG-HMK-IL-15 is a Biologically Active Growth Factor thatRequires Expression of IL-2Râ

[0126] In the next series of experiments, the FLAG-HMK-IL-15 fusionprotein was tested in order to determine whether it could function as abiologically active growth factor. PHA-activated human PBMCs proliferatein response to either FLAG-HMK-IL-15 or human recombinant IL-2, asdetected via the [³H]-TdR incorporation assay described above. A FLAGpeptide lacking the IL-15 sequence does not stimulate cell proliferation(FIG. 3a). As does IL-2, the FLAG-HMK-IL-15 fusion protein stimulatesproliferation of IL-2Rã⁺BAF-BO3 cell transfectants that express theIL-2Râ subunit (FIG. 3b, right-hand panel). The FLAG-HMK-IL-15 fusionprotein does not, however, stimulate the proliferation of parentalBAF-BO3 cells that were transfected with a vector lacking IL-2Râ chainsequences (FIG. 3b, left-hand panel). Thus, FLAG-HMK-IL-15 is abiologically active growth factor that requires expression of IL-2Râchains upon target cells in order to stimulate cellular proliferation.

[0127] Mitogen-Activated, but not Resting, PBMCs Express the IL-15RáSubunit

[0128] The FLAG-HMK-IL-15 fusion protein, biotinylated anti-FLAGantibody, and streptavidin-RED670 were employed to detect expression ofIL-15 binding sites on human PBMCs by cytofluorometric analysis. ThePBMCs tested were either freshly isolated or PHA-activated. These cellswere washed and incubated with either medium alone or FLAG-HMK-IL-15followed by anti-FLAG biotinylated Ab and streptavidin-RED670. Thestained cells were analyzed by flow cytometry (FIG. 4A). PBMCs that wereactivated with PHA expressed IL-15Rá proteins but resting PBMCs did not.In keeping with the result of the cross-linking experiments describedabove (FIG. 2), binding of FLAG-HMK-IL-15 to PHA activated PBMCs isblocked by a molar excess of rIL-15 (FIG. 4B), thereby demonstrating thespecificity of FLAG-HMK-IL-15 binding for IL-15 binding sites. Bothactivated CD4⁺ and CD8⁺ cells express IL-15á chains (FIG. 4C).Activation induced IL-15Rá chains were also detected on CD14⁺(monocyte/macrophage) cells and CD16⁺ (natural killer) cells.

[0129] IL-2Rá and IL-2Râ Subunits are not Required for IL-15 Binding

[0130] FACS analysis of PHA-activated PBMCs stained with FLAG-HMK-IL-15proteins and anti-CD25 Mab, against the IL-2Rá subunit, reveals cellpopulations expressing both IL-15Rá and IL-2Rá subunits, as well as cellpopulations that express either subunit, but not both (FIG. 5A). Thereare IL-2Rá⁺ cells that do not bind FLAG-HMK-IL-15 (FIG. 5A). Almost allPBMCs that were stimulated with PHA for only one day express eitherIL-15Rá or IL-2Râ chains, but not both proteins (FIG. 5B). In contrast,3 days following PHA stimulation, a far larger population of IL-15Rá⁺,IL-2Râ⁺ cells (double positive) and a far smaller population ofIL-15Rá⁺, IL-2Râ⁻ cells (single positive) were noted (FIG. 5B).Interestingly, there are IL-2Râ⁺ cells that fail to bind IL-15 (FIG.5B). Therefore, expression of IL-2Râ chains is not sufficient for IL-15binding.

[0131] Taken together, these data indicate that IL-15 can bind IL-15Rá⁺,IL-2Rá⁻, IL-2Râ⁻ cells. A similar conclusion was reached throughexperimentation that probed the interaction of IL-15 with IL-2Rá⁻, â⁻cells transfected with IL-15Rá subunit (Anderson et al., J. Biol. Chem.270:29862, 1995; Giri et al., EMBO J. 14:3654, 1995).

[0132] In addition to the requirement for IL-15Rá subunit expression,the IL-2Râ and IL-2Rã subunits are required to render cells sensitive toIL-15 triggered growth (FIG. 3, see also FIG. 5).

[0133] Dexamethasone, but not Cyclosporine or Rapamycin BlocksMitogen-induced Expression of the IL-15Rá Subunit

[0134] The effects of several immunosuppressive drugs on PHA-inducedexpression of IL-15Rá by PBMCs were also studied. The drugs tested werecyclosporine-A (CsA), rapamycin, and dexamethasone. The addition of CsAto PBMCs cultured with PHA resulted in a 20% reduction in the expressionof IL-2Rá, but IL-15Rá was unaffected (FIG. 6). The addition ofrapamycin actually resulted in enhanced IL-15Rá expression (FIG. 6C). Incontrast to the effects of CsA and rapamycin, dexamethasone powerfullyblocked the expression of IL-15Rá chains on mitogen activated PBMCs(FIG. 6D). In accordance with these data, PHA-activated,dexamethasone-treated cells proliferated poorly in response to IL-15,while the response to IL-2 was not inhibited by dexamethasone (FIG. 7).In this experiment, PMBCs were cultured in the presence of PHA andvarious immuno-suppressive drugs, and then washed and cultured furtherin the presence of medium alone (“none”), PHA, IL-2, or IL-15 for 2 daysbefore a [³H]-TdR pulse. The potent response of PHA-activated,dexamethasone-treated PBMCs to IL-2 proves that the ability ofdexamethasone to inhibit the proliferative response to IL-15 is not dueto a non-specific toxic effect.

[0135] The studies described above used FLAG-HMK-IL-15 and flowcytometry analysis to determine whether resting and mitogen-activatedPBMCs express the IL-15Rá subunit. The IL-15Rá subunit is rapidlyexpressed by activated but not resting lymphocytes, NK-cells, andmacrophages (FIG. 4).

[0136] These studies have also shown that the induction of IL-15Rá bymitogen is sensitive to an immunosuppressant: PBMCs pre-treated withdexamethasone and stimulated with PHA do not express IL-15Rá asvigorously as cells that were not pretreated with dexamethasone (FIG.6). Similarly, PBMCs pre-treated with dexamethasone do not proliferateas robustly in response to exogenously added IL-15 (FIG. 7). The cellsdo respond partially to IL-15. This may reflect that fact that IL-15Ráis expressed within 24⁺ hours of dexamethasone withdrawal. CsA, a potentinhibitor of IL-2 and IL-2Rá expression (Farrar et al., J. Biol. Chem.264:12562, 1989), is ineffective in preventing the mitogen-inducedexpression of the IL-15Rá chain (FIG. 6). There was a slight reductionin the proliferative response to exogenously added IL-15 among cellspre-treated with CsA for 72 hours (FIG. 7), however this reduction maybe due to inhibition of any number of events in the CsA-sensitive,Ca²⁺-dependent pathway of T cell activation. Since the same modestreduction in cell proliferation was observed in response to IL-2, CsAmay exert certain long lasting effects by blocking signaling pathway(s)shared by IL-2 and IL-15. These results, in conjunction with the datashowing CsA-independent induction of IL-15 gene expression, suggest thatCsA may not block IL-15 triggered immune responses. On the contrary,efficiently blocking the induction of the IL-15Rá subunit withdexamethasone may be very helpful in preventing IL-15 induced cellularproliferation.

[0137] In summary, the experiments presented above have demonstratedthat: (i) IL-15Rá subunits are rapidly expressed by activatedmacrophages, T cells, and NK cells, and (ii) induction of the IL-15Rásubunit is blocked by dexamethasone but not by CsA or rapamycin. Inaddition, the experiments have confirmed that the IL-15Rá subunit isnecessary and sufficient for IL-15 binding and that the FLAG-HMK-IL-15fusion protein is an extremely useful tool for studying IL-15 receptors.

A Comparative Analysis of the IL-2 and IL-15 Signalling Pathways

[0138] In this series of experiments, the intracellular signallingpathway that is initiated when IL-2 binds to the IL-2Rá, −â, −ã receptorcomplex was compared to the pathway initiated when IL-15 binds to thereceptor complex composed of IL-15Rá, IL-2Râ, and IL-2Rã.

[0139] The methods, in addition to those described above, required toperform these experiments follow.

[0140] Cell Culture, Cell Lysis and Protein Immunoblotting

[0141] Fresh human PBMCs were isolated as described above and culturedin RPMI-1640 medium supplemented with 10% FCS, penicillin, andstreptomycin (also as above). To achieve activation by a mitogen, PHA(10 ig/ml) was added to the culture for 3 days. PHA-stimulated PBMCswere washed three times with PBS, cultured for 14 hours in RPMI-1640medium containing 10% FCS, penicillin, and streptomycin, then culturedin medium containing recombinant human IL-2 (100 U/ml), recombinanthuman IL-15 (10 ng/ml) or non-supplemented medium, for 10 minutes at37EC.

[0142] After stimulation with interleukins, the cells were washed incold PBS and lysed in an ice-cold lysis buffer (150 mM NaCl, 20 mM Tris(pH 7.5), 1 mM phenymethylsulfonyl fluoride, 0.5 mg/ml leupeptin, 10mg/ml aprotinin, 1 mM Na₃VO₄, 50 mM NaF, and 1% NP-40). Lysates wereincubated for 15 minutes on ice and then pelleted at 100,000×g for 30minutes.

[0143] Soluble proteins were separated by SDS-PAGE (10% acrylamide)followed by transfer to a PVDF membrane (Millipore, Bedford, Mass.). Themembrane was then incubated in blocking buffer (25 mM HEPES (pH 7.4),150 mM NaCl, 0.05% Tween, and 2% BSA) for 2 hours at room temperature,followed by incubation with an anti-phosphotyrosine antibody, RC-20,conjugated to alkaline phosphate (Signal Transduction Labs, Inc.Lexington, Ky.) for 2 hours at room temperature. Washed blots weredeveloped in the BCIP/NBT phosphatase substrate solution forcalorimetric analysis (Kirkegard and Perry Laboratories, Inc.Gaithersburg, Md.).

[0144] The pattern of Tyrosine Phosphorylated Proteins Induced by IL-2is the Same as that Induced by IL-15 To select a population ofactivated, IL-2 responsive T cells, PBMCs were stimulated with PHA for 3days, washed, and then stimulated with IL-2 or IL-15 for an additional 2days. Cultivation of lymphocytes in IL-2 rich medium leads topropagation of lymphocytes that express a high copy number of thetri-molecular high-affinity IL-2R complex (Maslinski et al., J. Biol.Chem. 267:15281, 1992). Furthermore, cultivation of PHA-treated PBMCswith IL-2 or IL-15 propagates cells that are equally sensitive tofurther stimulation with either cytokine. Hence, according to the schemeshown in FIG. 8A, stimulation of mitogen-activated PBMCs with IL-2 orIL-15 does not select for a bulk cell population that then responds onlyto IL-2 or only to IL-15.

[0145] Further experiments were carried out using T cells pre-stimulatedwith PHA for 3 days followed by stimulation with IL-2 for 2 days. IL-2stimulated cell proliferation is a protein tyrosine kinase dependentevent (Maslinski et al. J. Biol. Chem. 267:15281, 1992); Remillard etal., J. Biol. Chem. 266:14167, 1991). Thus, an inhibitor of tyrosinekinases, genistein, was used to test whether the proliferative signalsinduced by IL-15 are mediated by the same tyrosine kinases as are theproliferative signals induced by IL-2. IL-2 and IL-15 both induce T cellproliferation and both manifest a similar dose-related sensitivity togenistein (FIG. 9).

[0146] To rule out the possibility that genistein was functioning as ageneral toxin, rather than a selective inhibitor of proteinkinase-dependent IL-2 or IL-15-induced cell proliferation, a standardtrypan blue exclusion assay was performed. Cell viability after 3 daysof incubation in the presence of genistein (10 Fg/ml) was 65% ″ 15% ofthe control (where no genistein was added). Therefore, most of theobserved inhibitory effect of genistein is due to inhibition of IL-2 andIL-15 triggered signal transduction.

[0147] Tyrosine phosphorylation events are critical for IL-15 stimulatedproliferation. Indeed, a direct comparison of the pattern of tyrosinephosphorylation shows that the protein phosphorylation induced by IL-2is identical to the protein phosphorylation induced by IL-15 (FIG. 10).This indicates that both cytokines stimulate similar, if not identical,protein tyrosine kinases.

[0148] IL-2 and IL-15 Stimulated Proliferative Events are EquallySensitive to Inhibition by Rapamycin

[0149] To further characterize the signaling events that are mediated byIL-2 and IL-15, the sensitivity of IL-2 and IL-15 induced T cellproliferation to rapamycin, which inhibits IL-2R signal transduction.Rapamycin, a macrolide with potent immunosuppressive activity (Sigal etal., Annu. Rev. Immunol. 10:519, 1992; Sehgal et al., ASHI Quarterly15:8, 1991), inhibits IL-2 induced signal transduction prior to or atthe level of activation of p70 S6 kinase and cyclin E dependent cdk2kinase (Price et al., Science 257:973, 1992; Bierer et al., Proc. Natl.Acad. Sci. USA 87:9231, 1990; Calvo et al., Proc. Natl. Acad. Sci. USA89:7571, 1992; and Chung et al., Cell 69:1227, 1992). PHA-stimulated,IL-2-activated cells were washed prior to incubation with rapamycin andcultured with IL-2 or IL-15. Again, the similarity of IL-2 and IL-15induced T cell proliferative events was evident: the proliferativeevents stimulated by these two interleukins were equally dose-sensitiveto inhibition by rapamycin (FIG. 10). Just as in studies involvinggenistein (see above), the possibility that rapamycin was toxic tolymphocytes was ruled out by performing standard trypan blue exclusionassays (Current Protocols in Immunology, Vol. 3, ed. Coico, R. New York:John Wiley & Sons, 1995). Cell viability after three days of incubationwith rapamycin (10 ng/ml) was 8.5% ″ 10% of the control, where no drugwas added. Therefore, the observed inhibition of cellular proliferationby rapamycin is due to an inhibition of IL-2 and IL-15 induced signaltransduction.

[0150] The Immunosuppressant Cyclosporin does not Inhibit Either IL-2 orIL-15 Induced Cellular Proliferation

[0151] The effect of a second immunosuppressant, cyclosporine-A (CsA),on IL-15 induced cell proliferation was also examined. Cyclosporine-A isa peptide that forms complexes with cyclophilin; these complexes bind toand inhibit the enzymatic activity of the cellular phosphatecalcineurin, resulting, among other effects, in blockade of IL-2 andIL-2Rá gene transcription (Sigal et al., Annu. Rev. Immunol. 10:519,1992). T cells expressing the tri-molecular IL-2R complex and stimulatedwith exogenously added IL-2 are not sensitive to CsA (Sigal et al.,Annu. Rev. Immunol. 10:519, 1992). It has now been shown that CsA doesnot inhibit IL-15 or IL-2 triggered cellular proliferation (FIG. 11),again revealing similarities between IL-2 and IL-15 induced cellularproliferation.

[0152] The doses of CsA used in these studies (to block PHA-induced IL-2gene expression) were tested in order to provide evidence that asufficient amount of CsA was being used. CsA (100 ng/ml) completelyblocked PHA-induced IL-2 mRNA expression as judged by the results ofRT-PCR and competitive PCR techniques, as described before (Lipman etal., J. Immunol. 152:5120, 1994). This result confirms the efficacy ofCsA in the culture system used herein. Moreover, it discounts thepossibility that IL-15 induces IL-2 gene expression that, in turn, couldbe responsible for T cell proliferation.

[0153] The IL-2Râ Subunit is Critical for both IL-2 and IL-15 SignalTransduction

[0154] The hypothesis that elements of the IL-2Râ chain that arerequired for IL-2 signal transduction are also required for IL-15 signaltransduction was tested. Previous studies indicated that BAF-BO3 cellsthat express endogenous IL-2Rá and IL-2Rã subunits can be rendered IL-2dependent upon transfection with cDNA encoding human the IL-2Râ protein(Hatakeyama et al., Science 244:551, 1989). In contrast, BAF-BO3 cellsthat express a mutant IL-2Râ protein, which lacks a 71 amino acidserine-rich region, do not respond to IL-2 (Hatakeyama et al., Science252:1523, 1991; Hatakeyama et al., Science 244:551, 1989; and Fung etal., J. Immunol. 147:1253, 1991). Therefore, the ability of IL-15 toinduce the proliferation of IL-2Ráã⁺ BAF-BO3 cells that express eitherthe wild-type IL-2Râ subunit or a mutant IL-2Râ protein that lacks theserine rich region (S⁻). While both IL-2 and IL-15 stimulate theproliferation of cells that express the wild-type IL-2Râ subunit,neither cytokine is able to stimulate IL-2Ráã⁺ BAF-BO3 cells thatexpress mutant S⁻ IL-2Râ subunit (FIG. 12). These results demonstratethat the serine-rich region of the IL-2Râ subunit is critical for bothIL-2 and IL-15 triggered signal transduction.

[0155] The serine-rich region of the IL-2Râ subunit binds severaltyrosine kineses including Jak-1 and Syk (Minami et al., Immunity 2:89,1995). The same kinases may play a critical role in both IL-2 and IL-15signal transduction. Since BAF-BO3 cells cannot express IL-2 (Andersonet al., J. Biol. Chem. 270:29862, 1995), even upon stimulation withIL-15, these results indicate that IL-15 induced cellular proliferationdoes not require induction of IL-2 gene expression.

[0156] The experiments performed in this series (see also Lin et al.,Immunity 2:331, 1995; Anderson et al., J. Biol. Chem. 270:29862, 1995)indicate that IL-2 and IL-15 triggered signal transduction useoverlapping, perhaps identical, signaling pathways, and that agents thatblock IL-2 signaling are highly likely to block IL-15 signaling.Although these experiments compared relatively early events in thecourse of IL-2 and IL-15 intracellular signal transduction, the terminalphase of IL-2 and IL-15 signal transduction are also likely to be quitesimilar insofar as activation of target cells with IL-2 or IL-15 givesrise to expression of the same DNA binding proteins (Giri et al., EMBOJ. 13:2822, 1994). These results also suggest that the function of theIL-15Rá and IL-2Rá chain are similar, i.e., that they are most importantfor cytokine binding affinity and have a negligible role in signaltransduction.

[0157] Decreasing the viability of activated T cells or blocking thesignal transduction pathways activated by IL-2 and IL-15 provides a wayto decrease the production of lymphokines and mitogens that contributeto accelerated atherosclerosis, allograft rejection, certain leukemiasand other immune-mediated pathologies. When activated, T cellsproliferate and express receptors on their cell surface forinterleukins. In addition, activated T cells release at least threelymphokines: gamma interferon, B cell differentiation factor II, andIL-3. These lymphokines can produce various undesirable events, such asallograft rejection. In contrast, resting T cells and long-term memory Tcells do not express lymphokine receptors. This difference in receptorexpression provides a means to target activated immune cells withoutinterfering with resting cells. Molecules designed to recognize somesubunit of the IL-15R will recognize activated monocytes/macrophages aswell as activated T cells and can be used to selectively inhibit ordestroy these cells. Derivatives of IL-15 that bind to an IL-15R subunitbut that lack IL-15 activity, either because they block the bindingand/or uptake of bona fide IL-15, are useful in the method of theinvention. The mutant IL-15 molecule described below provides a workingexample of such a derivative.

A Mutant IL-15 Polypeptide that Functions as an Antagonist of Wild-TypeIL-15

[0158] Genetic Construction of Mutant IL-15

[0159] The human IL-15 protein bearing a double mutation (Q149D; Q156D)was designed to target the putative sites critical for binding to theIL-2Rã subunit. The polar, but uncharged glutamine residues at positions149 and 156 (FIG. 12) were mutated into acidic residues of aspartic acid(FIG. 13) utilizing PCR-assisted mutagenesis. A cDNA encoding the doublemutant of IL-15 was amplified by PCR utilizing a synthetic senseoligonucleotide [5′-GGAATTCAACTGGGTGAATGTAATA-3′(SEQ ID NO: 1); EcoRIsite (underlined hexamer) plus bases 145-162] and a synthetic antisenseoligonucleotide[5′-CGGGATCCTCAAGAAGTGTTGATGAACATGTCGACAATATGTACAAAACTGTCCAAAAA T-3′(SEQ ID NO: 3); BamHI site (underlined hexamer) plus bases 438-489;mutated bases are singly underlined]. The template was a plasmidcontaining cDNA that encodes human FLAG-HMK-IL-15. The amplifiedfragment was digested with EcoRI/BamHI and cloned into the pAR(DRI)59/60 plasmid digested with EcoRI/BamRI as described (LeClair et al.,Proc. Natl. Acad. Sci. USA 89:8145, 1989). The presence of a mutation atresidue 156 was confirmed by digestion with SalI; the mutationintroduces a new SalI restriction site. In addition, mutations wereverified by DNA sequencing, according to standard techniques. TheFLAG-HMK-IL-15 (Q149D; Q156D) double mutant protein was produced,purified, and verified by sequencing as described above for theFLAG-HMK-IL-15 wild-type protein.

[0160] Using this same strategy, mutants that contain only a singleamino acid substitution, either at position 149 or at position 156 wereprepared. As described above, these positions (149 and 156) correspondto positions 101 and 108, respectively, in the mature IL-15 polypeptide,which lacks a 48-amino acid signal sequence.

[0161] Similarly, this strategy can be used to incorporate any otheramino acid in place of the glutamine residues at positions 149 or 156 orto introduce amino acid substitutions at positions other than 149 and/or156.

[0162] Proliferation of BAF-BO3 Cells in the Presence of IL-15 RelatedProteins

[0163] In order to study the effect of various IL-15 related proteins,including the mutant polypeptides described above, on the proliferationof BAF-BO3 cells, the following experiment was performed in vitro.BAF-BO3 cells were transfected in culture with DNA from eitherpRcCMV-IL-2Râ or pRcCMV-0. These plasmids differ in that pRcCMV-IL-2Râcontains an insert encoding the human IL-2Râ subunit and pRcCMV-0 doesnot. Following transfection, the cells were washed and treated witheither: (1) unsupplemented medium (“none”), (2) IL-2, (3) IL-3 rich WEHIcell supernatant (WEHI), (4) FLAG-HMK-IL-15, (5) FLAG-HMK-IL-15 Q149Dsingle mutant (149), (6) FLAG-HMK-IL-15 Q156D double mutant (DM), (7)IL-15, or (8) IL-15+FLAG-HMK-IL-15 Q149D Q156D double mutant (FIG. 15).Cells transfected with pRcCMV-0 DNA, did not proliferate in response toany stimulus except WEHI cell supernatant. In contrast, followingtransfection with pRcCMV-IL-2Râ DNA, IL-2, WEHI cell supernatant,FLAG-HMK-IL-15, FLAG-HMK-IL-15 Q149D single mutant, and IL-15 stimulatedcellular proliferation. The IL-2Râ expressing cells did not proliferatein response to the double mutant IL-15. The double mutant IL-15polypeptide may inhibit BAF-BO3 proliferation in a dose-dependentmanner: addition of 30 il (approximately 50 ig/ml) of the double mutantIL-15 inhibited proliferation more completely than did addition of 20 iLof the same concentration of the double mutant IL-15.

[0164] Proliferation of PHA-Stimulated Human PBMCs in the Presence ofIL-15 Related Proteins

[0165] Human PBMCs prestimulated with PHA (2 ig/ml) for 72 hours werewashed and cultured in the presence of IL-15 related proteins including:(1) the IL-15 double mutant, FLAG-HMK-IL-15-Q149D-Q156D, (2) the IL-15single mutant, FLAG-HMK-IL-15-Q149D, or (3) the IL-15 single mutant,FLAG-HMK-IL-15-Q156D (FIG. 16). Medium without an IL-15 relatedpolypeptide served as a control. The proliferative response was thenassessed.

[0166] FACS Analysis of PHA-Activated Human PBMCs Stained withFLAG-HMK-IL-15-Double Mutants

[0167] The ability of the FLAG-HMK-IL-15 double mutant polypeptide tobind PHA-activated human PBMCs was demonstrated as follows.PHA-activated PBMCs were washed and incubated with medium alone, or withthe FLAG-HMK-IL-15 double mutant. The cells were then incubated with ananti-FLAG biotinylated antibody and stained with streptavidin conjugatedto RED670. The stained cells were analyzed by flow cytometry (FIG. 17).

[0168] FACS Analysis of Leukemic Cell Lines Stained with Wild-TypeFLAG-HMK-IL-15

[0169] In a series of experiments similar to those above, the ability ofthe wild-type FLAG-HMK-IL-15 polypeptide to bind leukemia cells wasshown. The cells treated were obtained from the leukemic cell linesMOLT-14, YT, HuT-102, and from cell lines currently being established atBeth Israel Hospital (Boston, Mass.), and named 2A and 2B. The culturedcells were washed and incubated with either medium alone or with mediumcontaining the FLAG-HMK-IL-15 wild-type polypeptide (FIG. 18). The cellswere then incubated with the biotinylated anti-FLAG antibody and stainedwith RED670-conjugated streptavidin. The stained cells were analyzed byflow cytometry.

[0170] Genetic Construction of Additional Mutant IL-15 ChimericPolypeptides

[0171] In addition to the FLAG-HMK-IL-15 chimera, which provides themutant IL-15 with an antigenic tag, numerous other polypeptides can belinked to any mutant of IL-15. For example, mutant IL-15 can be linkedto serum albumin or to the Fc fragment of the IgG subclass ofantibodies, according to the following method.

[0172] Genetic Construction of Mutant IL-15/Fc−−

[0173] cDNA for Fcã2a can be generated from mRNA extracted from an IgG2asecreting hybridoma using standard techniques with reverse transcriptase(MMLV-RT; Gibco-BRL, Grand Island, N.Y.) and a synthetic oligo-dT(12-18) oligonucleotide (Gibco BRL). The mutant IL-15 cDNA can beamplified from a plasmid template by PCR using IL-15 specific syntheticoligonucleotides.

[0174] The 5′ oligonucleotide is designed to insert a unique NotIrestriction site 40 nucleotides 5′ to the translational start codon,while the 3′ oligonucleotide eliminates the termination codon andmodifies the C-terminal Ser residue codon usage from AGC to TCG toaccommodate the creation of a unique BamHI site at the mutant IL-15/Fcjunction. Synthetic oligonucleotides used for the amplification of theFcã2a domain cDNA change the first codon of the hinge from Glu to Asp inorder to create a unique BamHI site spanning the first codon of thehinge and introduce a unique XbaI site 3′ to the termination codon. TheFc fragment can be modified so that it is non-lytic, i.e., not able toactivate the complement system. To make the non-lytic mutant IL-15construct (mIL-15/Fc−−), oligonucleotide site directed mutagenesis isused to replace the C′1q binding motif Glu318, Lys320, Lys322 with Alaresidues. Similarly, Leu235 is replaced with Glu to inactivate the FcãRI binding site. Ligation of cytokine and Fc″ components in the correcttranslational reading frame at the unique BamHI site yields a 1,236basepair open reading frame encoding a single 411 amino acid polypeptide(including the 18 amino acid IL-15 signal peptide) with a total of 13cysteine residues. The mature secreted homodimeric IL-15/Fc−−ispredicted to have a total of up to eight intramolecular and threeinter-heavy chain disulfide linkages and a molecular weight ofapproximately 85 kDa, exclusive of glycosylation.

[0175] Expression and Purification of mIL-15 Fc Fusion Proteins

[0176] Proper genetic construction of both mIL-15/Fc++, which carriesthe wild-type Fcã2a sequence, and mIL-15/Fc−−can be confirmed by DNAsequence analysis following cloning of the fusion genes as NotI-XbaIcassettes into the eukaryotic expression plasmid pRc/CMV (Invitrogen,San Diego, Calif.). This plasmid carries a CMV promoter/enhancer, abovine growth hormone polyadenylation signal, and a neomycin resistancegene for selection with G418. Plasmids carrying the mIL-15/Fc++ormIL-15/Fc−−fusion genes are transfected into Chinese hamster ovary cells(CHO-K1, available from the American Type Culture Collection) byelectroporation (1.5 kV/3 iF/0.4 cm/PBS) and selected in serum-freeUltra-CHO media (BioWhittaker Inc., Walkerville, Md.) containing 1.5mg/ml of G418 (Geneticin, Gibco BRL). After subcloning, clones thatproduce high levels of the fusion protein are selected by screeningsupernatants from IL-15 by ELISA (PharMingen, San Diego, Calif.).mIL-15/Fc fusion proteins are purified from culture supernatants byprotein A sepharose affinity chromatography followed by dialysis againstPBS and 0.22 im filter sterilization. Purified proteins can be storedat−20EC before use.

[0177] Western blot analysis following SDS-PAGE under reducing (withDTT) and non-reducing (without DTT) conditions can be performed usingmonoclonal or polyclonal anti-mIL-15 or anti Fcã primary antibodies toevaluate the size and isotype specificity of the fusion proteins.

[0178] Standardization of the Biological Activity of Recombinant MutantIL-15 and mIL-15/Fc−−Proteins

[0179] Using the RT-PCR strategy and 5′ NotI sense oligonucleotideprimer described above, mutant IL-15 cDNA with an XbaI restriction siteadded 3′ to its native termination codon, can be cloned into pRc/CMV.This construct is then transiently expressed in COS cells (availablefrom the American Type Culture Collection). The cells may be transfectedby the DEAE dextran method and grown in serum-free UltraCulture? media(BioWhittaker Inc.). Day 5 culture supernatant is sterile filtered andstored at−20EC for use as a source of recombinant mutant IL-15 protein(rmIL-15).

[0180] Mutant IL-15/Fc−−and rmIL-15 mutant protein concentrations can bedetermined by ELISA as well as by bioassay, as described(Thompson-Snipes et al., J. Exp. Med. 173:507, 1991).

[0181] The functional activity of mutant IL-15/Fc−−can be assessed by astandard T cell proliferation assay, as described above.

[0182] Determination of mIL-15/Fc−−or mIL-15/Fc++

[0183] Circulating Half-life

[0184] Serum concentration of mIL-15/Fc or mIL-15/Fc++fusion proteinscan be determined over time following a single intravenous injection ofthe fusion protein. Serial 100 il blood samples can be obtained bystandard measures at intervals of 0.1, 6, 24, 48, 72, and 96 hours afteradministration of mutant IL-15/Fc−−protein. Measurements employ an ELISAwith a mIL-15 mAb as the capture antibody and horseradish peroxidaseconjugated to an Fc″2a mAb as the detection antibody, thus assuring thisassay is specific for only the mutant IL-15/Fc−−.

IL-15 Mutants in the Treatment of Arthritis

[0185] Rheumatoid arthritis (RA) is a T cell dependent autoimmunedisease in which mononuclear cells infiltrate the joints, causinginflammation and progressive destruction of articular cartilage andbone. Therapies for alleviating RA have been tested in the murine typeII collagen-induced arthritis (CIA) model (see, e.g., Courtenay et al.,Nature 283:666-668, 1980).

[0186] Direct contact between collagen type II (CII) immune T cells andmonocytes or T cells and synoviocytes leads to the production ofproinflammatory cytokines, such as IL-1â and TNF-á (Burger et al.,Arthritis and Rheumatism 41:1748-1759, 1998; Rezzonico et al., J. Biol.Chem. 30:18720-18728, 1998; Vey et al., J. Immunol. 149:2040-2046, 19??;Isler et al., Eur. Cytokine. Netw. 4:15-23, 1993) and metalloproteinases(Miltenburg et al., J. Immunol. 154:2655-2667, 1995). TNFá is stronglyimplicated in the pathogenesis of CIA and clinical RA. In patients withRA, T cells that infiltrate synovia, monocytes, and macrophages producehigh levels of TNFá (Akatsuka et al., Microbiol. Immunol. 41:367-370,1997; Feldmann et al., Ann. Rev. Immunol. 14:397-440, 1996). The successof therapeutic strategies that neutralize TNFá in the murine CIA modeland in clinical RA also indicates that TNFá plays a crucial role in thepathogenesis of arthritis (Knight et al., Mol. Immunol. 30:1443-1453,1993; Wooley et al., J. Immunol. 151:6602-6607, 1993; Williams et al.,Immunol. 84:433-439, 1995; Elliott et al., Lancet 344:1105-1110, 1994;Elliott et al., Lancet 344:1125-1127, 1994; Moreland et al., N. Engl. J.Med. 337:141-147, 1997).

[0187] We have found that DBA/1 mice treated with an IL-15 mutant/Fcã2aprotein, which acts in part as a long lived, high affinity IL-15receptor (IL-15R) antagonist, have a markedly decreased incidence ofRA-like collagen type II arthritis. In addition, those mice that dodevelop arthritis experience markedly less intense symptoms.Surprisingly, the benefits of IL-15R antagonists persist long after thetreatment is discontinued. As shown by the experiments described below,expression of the proinflammatory cytokines IL-1â and TNFá wasdramatically decreased in IL-15 mutant/Fcã2a-treated mice, andhistological analyses confirmed that treatment with IL-15 mutant/Fcã2aprotects joints from leukocytic infiltration. These results support thehypothesis that IL-15 and IL-15R-positive mononuclear leukocytes play amajor role in the inflammatory processes that cause arthritis anddemonstrate the efficacy of IL-15R antagonists in treating thiscondition.

[0188] Expression and Purification of IL-15 mutant/Fcã2c Protein

[0189] NS.1 cells, a B cell line available from the American TypeCulture Collection, were transfected with a plasmid carrying a fusiongene encoding human IL-15 linked to murine Fcã2a cDNA. The plasmid wasconstructed as described above. (see “Genetic Construction of mutantIL-15”) The transfected cells were cloned and cultured in serum-freeUltraculture medium (BioWhittaker Inc., Walkersville, Md.) containing100 ig/ml Zeocin (Invitrogen, San Diego, Calif.). IL-15 mutant/Fcã2afusion proteins were purified from the culture supernatant from highproducing clones by protein A-Sepharose affinity chromatography(Pharmacia, Piscataway, N.J.). The expressed protein was dialyzedagainst PBS and sterilized by passage through a 0.22 im filter. Purifiedprotein was stored at −20EC.

[0190] Induction of Collagen-induced Arthritis

[0191] Male DBA/1 mice, 8-9 weeks old, were obtained from The JacksonLaboratory (Bar Harbor, Me.) and used in this series of experiments. CII(1 mg) derived from chicken sternal cartilage (Sigma Chemical Co., St.Louis, Mo.) was dissolved by placing it in 0.1 M acetic acid (1 ml) at4EC for 24 hours and then emulsified with an equal volume of Freund'scomplete adjuvant (CFA; Sigma Chemical Co., St. Louis, Mo.; see,Courtenay et al., Nature 283:666-668, 1980). To induce CIA, 300 il ofthe emulsion was injected intradermally at the base of the tail of DBA/1mice at day 0. On day 21, the animals received a second injection; 200ig of CII was injected intraperitoneally.

[0192] Treatment for Arthritis

[0193] Twenty-one days following the last injection of CII, animals weredivided into two groups. The first group received daily intraperitonealinjections of IgG2a (Becton Dickinson, San Jose, Calif.) at aconcentration of 1.5 ig/mouse, and the second group received dailyintraperiotoneal injections of IL-15 mutant/Fcã2a protein at a finalconcentration of 1.5 ig/mouse. Treatment was continued until animalswere sacrificed or a maximum of 10 days.

[0194] Mice were evaluated every day for arthritis based on thefollowing arthritis severity index: grade 0—no swelling; grade 1—mildswelling or erythema; grade 2—pronounced edema or redness of the paw orof several digits; grade 3—severe swelling of the entire paw orankylosis. All four limbs of each animal were graded, resulting in amaximum clinical score of 12/animal.

[0195] Six animals were sacrificed 21 days after immunization. NineteenIgG2a-treated and eighteen IL-15 mutant/Fcã2a-treated mice weresacrificed 4 days, 7 days, and 21 days after the onset of arthritis (25,28, and 42 days post-immunization, respectively). All remaining IgG2a-and IL-15 mutant/Fcã2a-treated mice were sacrificed on day 42.

[0196] Histology

[0197] The animals' paws were removed post-mortem, fixed in 1%paraformaldehyde for three days, and then decalcified in 5% EDTA for twoweeks. The tissue was then embedded in paraffin and sectioned. Sagittalserial sections were stained with hematoxylin and eosin beforeexamination.

[0198] Measurement of Serum Cytokines

[0199] Serum levels of TNFá and IL-1â were assayed by ELISA (R&DSystems, Minneapolis, Minn.) according to the manufacturer'sinstructions. Lower limits of detection were at 5-10 pg/ml for TNFá andat 10 pg/ml for IL-1â.

[0200] mRNA Analysis

[0201] Tissue samples were homogenized with a polytron (Kinematika,Switzerland), and total cellular RNA was extracted by RNASTAT 60? (TelTest, Friendswood, Tex.). The RNA samples were processed according tothe manufacturer's instructions. RT-PCR was performed as described inStrehlau et al. (Proc. Natl. Acad. Sci. USA 94:695-700, 1997). Briefly,1 ig of total RNA isolated from individual joint samples was reversetranscribed into cDNA utilizing M-MLV reverse transcriptase (Promega,Madison, Wis.). The cDNA was then amplified in a 25 il reaction volumecontaining 3 il of cDNA samples, 2.5 mM of each deoxynucleotidetriphosphate, 1.5 mM MgCl₂, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 5 U/ilTaq DNA polymerase. (Perkin Elmer, Roche Molecular Systems, Inc.,Branchburg, N.J.), and 100 ng of sense and antisense primers. Thespecific primers used for hybridization to murine INF-ã, IL-10, TCR Câand GAPDH cDNA, the latter as an internal control, were described inSteiger et al., (J. Immunol. 155:489-498, 1995). The primers used toamplify murine TNFá were 5′-CCAACGGCATGGATCTCAAAGAC-3′ (sense; SEQ IDNO: 9) and 5′-TCACTGTCCCAGCATCTTGTGTTTC-3′ (antisense; SEQ ID NO: 10).The primers used to amplify murine IL-1â were5′-TGCACTACAGGCTCCGAGATGAAC-3′ (sense; SEQ ID NO: 11) and5′-CATCAGAGGCAAGGAGGAAACACAG-3′ (antisense; SEQ ID NO: 12). The PCR wasperformed in the Gene Amp? PCR system 2400 (Perkin Elmer, Cetus,Norwalk, Conn.) under the following conditions: denaturation at 94EC for30 seconds; annealing at 60EC for INFã, at 62EC for IL-10, at 55EC forGAPDH, and at 59EC for TNFã and IL-1â; and extension at 72EC for 45seconds. Forty cycles were made. A negative control, the omission ofcDNA in the PCR reaction mixture, was included for each PCRamplification. After amplification, the samples (10 il) were separatedon ethidium bromide-containing 1.5% agarose gels. The DNA was visualizedand photographed using an ultraviolet transilluminator (Gel Doc 1000?,Bio Rad, Hercules, Calif.).

[0202] Measurement of Serum Anti-CII Antibody Levels

[0203] A sandwich ELISA was used to assess levels of serum specific IgG1and IgG2a to CII (see, Kageyama et al., J. Immunol. 161:1542-1548,1998). The serum samples were collected 21, 25, 28, and 42 days afterprimary immunization and frozen at −70EC. Native chicken CII wasdissolved in 0.1 M acetic acid at 1 mg/ml and diluted with 0.1 M sodiumbicarbonate at 0.1 ig/ml (pH 9.6). 96-well plates (Costar, Cambridge,Mass.) were coated with 100 il of CII Ag solution and incubatedovernight at 4EC. Plates were then washed three times with TBS (20 mMTris pH 8.0, 150 mM NaCl) containing 0.05% Tween 20 and subsequentlyblocked with 170 il of 10% BSA in TBS for 2 hours at room temperature.Plates were washed three times and then incubated with 100 il/well ofserum serially diluted in TBS-Tween 20/10% BSA for 1 hour at 37EC. Afterthree washes, 100 il of peroxidase conjugate (1:500 dilution) was addedto detect IgG1 subclass antibodies. The peroxidase conjugate was appliedfor 30 minutes at 37EC. After washing three times, 100 il ofO-phenylenediamine (0.5 mg/ml) dissolved in 0.1 M citric acid and 0.2 MNa₂HPO₄ containing 0.001% H₂O₂ was added. The reaction was stopped byaddition of 0.25 NH₂SO₄ (50 il/well). Quantitation was established by anELISA reader (at 490 nm).

[0204] Treatment with IL-15 Mutant/Fcã2a Delays the Onset and Diminishesthe Severity of CIA

[0205] DBA/1 mice immunized with an intradermal injection of CII andchallenged 21 days later with CII developed severe arthritis. Dailytreatment with the IL-15 mutant/Fcã2a protein for a maximum of 10 daysdelayed the onset of disease and decreased the severity and incidence ofCIA (relative to mice receiving an IgG control protein). Data analysiswas performed using a Kaplan-Meier cumulative plot for even free ofdisease and comparison between groups was performed using a logrank testfor event time (Statview 5.1, SAS Institute Inc., Cory, N.Y.) *p<0.01.

[0206] The severity of arthritis was monitored by a macroscopic score(and, as described above, was based on the following arthritis severityindex: grade 0—no swelling; grade 1—mild swelling or erythema; grade2—pronounced edema or redness of the paw or of several digits; grade3—severe swelling of the entire paw or ankylosis). A marked reduction inclinical score an in the number of arthritic paws was observedthroughout the treatment period. Remarkably, the beneficial effect ofthe IL-15 mutant/Fcã2a protein treatment persisted following cessationof treatment. Mice were killed at various times after the onset ofclinical arthritis, and the unaffected and affected paws were examined.Histological analyses showed that the marked tissue infiltration ofmononuclear cells observed in sections of IgG-treated mice was vastlydecreased in IL-15 mutant/Fcã2a-treated mice.

[0207] The Effect of IL-15 mutant/Fcã2a on Cytokine Expression in JointTissue.

[0208] The effect of IL-15 mutant/Fcã2a treatment on the expression ofTNFá and IL-1â was analyzed because these cytokines play a central rolein joint inflammation through the induction of other proinflammatorycytokines and metalloproteinases (Dayer et al., J. Exp. Med.162:2163-2168, 1985; MacNaul et al., J. Biol. Chem. 265:17238-17245,1990).

[0209] Mice immunized with CII and treated with either control IgG orIL-15 mutant/Fcã2a were sacrificed at day 25, day 28, day 42, or day 68.Expression of IL-1â and TNFá was examined at the transcriptional andtranslational levels. Sera were processed for protein quantification andjoints for mRNA analyses and immunostaining. IL-1â was detected in 40%of IgG-treated mice but in only 10% of IL-15 mutant/Fcã2a-treated mice.TNFá protein was never detected in the sera. In parallel, we assessedcytokine mRNA expression from joint tissues using RT-PCR andimmunostaining. Both TNFá and IL-1âare massively expressed in joints ofmice treated with control protein, but these cytokines were expressed inonly a minority of mice treated with the IL-15 related fusion protein.These data demonstrate that the IL-15 mutant/Fcã2a protein powerfullydiminishes expression of proinflammatory cytokines. Both IFNã and IL-10were expressed in some control-treated mice, but these cytokines weredetected in a minority of IL-15 antagonist-treated mice.

[0210] The Anti-CII Humoral Response is Not Altered by IL-15 Antagonist.

[0211] To determine whether treatment with IL-15 mutants inhibitsanti-CII antibody production, serum from both experimental and controlanimals was obtained 25, 28, and 42 days following immunization.Anti-CII antibody levels varied greatly between mice. The variation wasindependent of the day of collection and no significant difference inthe expression of IgG1 or IgG2a subtype antibodies was observed betweenthe two treatment groups. These data indicate that treatment with theIL-15 mutant/Fcã2a protein does not produce clinical improvement throughan effect upon anti-CII antibody production.

[0212] The Effect of IL-15 Mutant/Fcã2a on the Intraarticular T CellResponse.

[0213] To determine if the T cell response was altered by IL-15mutant/Fcã2a treatment, we analyzed TCR Câ gene expression in jointtissues of control-treated or IL-15 mutant-protein-treated mice byQRT-PCR. As the common chain of TCRâ is constitutively expressed by Tcells, a decrease in the number of T cells present in the joint shouldbe accompanied by a decrease in the abundance of TCR Câ gene transcriptswithin the tissue. The magnitude of intraarticular TCR Câ geneexpression is decreased in IL-15 mutant/Fcã2a-treated mice as comparedto control IgG-treated mice. These results indicate that treatment withIL-15 related fusion proteins inhibits:

[0214] (1) T cells infiltration of the inflammatory site or

[0215] (2) proliferation of T cells within the joint tissues.

1 12 1 25 DNA Artificial Sequence synthetically generated primer 1ggaattcaac tgggtgaatg taata 25 2 26 DNA Artificial Sequencesynthetically generated primer 2 cgggatcctc aagaagtgtt gatgaa 26 3 60DNA Artificial Sequence synthetically generated primer 3 cgggatcctcaagaagtgtt gatgaacatg tcgacaatat gtacaaaact gtccaaaaat 60 4 7 PRTArtificial Sequence synthetically generated peptide 4 Asp Tyr Lys AspAsp Asp Lys 1 5 5 489 DNA Homo sapiens CDS (1)...(486) 5 atg aga att tcgaaa cca cat ttg aga agt att tcc atc cag tgc tac 48 Met Arg Ile Ser LysPro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 ttg tgt tta cttcta aac agt cat ttt cta act gaa gct ggc att cat 96 Leu Cys Leu Leu LeuAsn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 gtc ttc att ttg ggctgt ttc agt gca ggg ctt cct aaa aca gaa gcc 144 Val Phe Ile Leu Gly CysPhe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 aac tgg gtg aat gta ataagt gat ttg aaa aaa att gaa gat ctt att 192 Asn Trp Val Asn Val Ile SerAsp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 caa tct atg cat att gat gctact tta tat acg gaa agt gat gtt cac 240 Gln Ser Met His Ile Asp Ala ThrLeu Tyr Thr Glu Ser Asp Val His 65 70 75 80 ccc agt tgc aaa gta aca gcaatg aag tgc ttt ctc ttg gag tta caa 288 Pro Ser Cys Lys Val Thr Ala MetLys Cys Phe Leu Leu Glu Leu Gln 85 90 95 gtt att tca ctt gag tcc gga gatgca agt att cat gat aca gta gaa 336 Val Ile Ser Leu Glu Ser Gly Asp AlaSer Ile His Asp Thr Val Glu 100 105 110 aat ctg atc atc cta gca aac aacagt ttg tct tct aac ggg aat gta 384 Asn Leu Ile Ile Leu Ala Asn Asn SerLeu Ser Ser Asn Gly Asn Val 115 120 125 aca gaa tct gga tgc aaa gaa tgtgag gaa ctg gag gaa aaa aat att 432 Thr Glu Ser Gly Cys Lys Glu Cys GluGlu Leu Glu Glu Lys Asn Ile 130 135 140 aaa gaa ttt ttg cag agt ttt gtacat att gtc caa atg ttc atc aac 480 Lys Glu Phe Leu Gln Ser Phe Val HisIle Val Gln Met Phe Ile Asn 145 150 155 160 act tct tga 489 Thr Ser 6162 PRT Homo sapiens 6 Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile SerIle Gln Cys Tyr 1 5 10 15 Leu Cys Leu Leu Leu Asn Ser His Phe Leu ThrGlu Ala Gly Ile His 20 25 30 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly LeuPro Lys Thr Glu Ala 35 40 45 Asn Trp Val Asn Val Ile Ser Asp Leu Lys LysIle Glu Asp Leu Ile 50 55 60 Gln Ser Met His Ile Asp Ala Thr Leu Tyr ThrGlu Ser Asp Val His 65 70 75 80 Pro Ser Cys Lys Val Thr Ala Met Lys CysPhe Leu Leu Glu Leu Gln 85 90 95 Val Ile Ser Leu Glu Ser Gly Asp Ala SerIle His Asp Thr Val Glu 100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn SerLeu Ser Ser Asn Gly Asn Val 115 120 125 Thr Glu Ser Gly Cys Lys Glu CysGlu Glu Leu Glu Glu Lys Asn Ile 130 135 140 Lys Glu Phe Leu Gln Ser PheVal His Ile Val Gln Met Phe Ile Asn 145 150 155 160 Thr Ser 7 489 DNAArtificial Sequence synthetically generated nucleic acid sequence 7 atgaga att tcg aaa cca cat ttg aga agt att tcc atc cag tgc tac 48 Met ArgIle Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 10 15 ttgtgt tta ctt cta aac agt cat ttt cta act gaa gct ggc att cat 96 Leu CysLeu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 25 30 gtc ttcatt ttg ggc tgt ttc agt gca ggg ctt cct aaa aca gaa gcc 144 Val Phe IleLeu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45 aac tgg gtgaat gta ata agt gat ttg aaa aaa att gaa gat ctt att 192 Asn Trp Val AsnVal Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60 caa tct atg catatt gat gct act tta tat acg gaa agt gat gtt cac 240 Gln Ser Met His IleAsp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80 ccc agt tgc aaagta aca gca atg aag tgc ttt ctc ttg gag tta caa 288 Pro Ser Cys Lys ValThr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 85 90 95 gtt att tca ctt gagtcc gga gat gca agt att cat gat aca gta gaa 336 Val Ile Ser Leu Glu SerGly Asp Ala Ser Ile His Asp Thr Val Glu 100 105 110 aat ctg atc atc ctagca aac aac agt ttg tct tct aac ggg aat gta 384 Asn Leu Ile Ile Leu AlaAsn Asn Ser Leu Ser Ser Asn Gly Asn Val 115 120 125 aca gaa tct gga tgcaaa gaa tgt gag gaa ctg gag gaa aaa aat att 432 Thr Glu Ser Gly Cys LysGlu Cys Glu Glu Leu Glu Glu Lys Asn Ile 130 135 140 aaa gaa ttt ttg gacagt ttt gta cat att gtc gac atg ttc atc aac 480 Lys Glu Phe Leu Asp SerPhe Val His Ile Val Asp Met Phe Ile Asn 145 150 155 160 act tct tga 489Thr Ser 8 162 PRT Artificial Sequence synthetically generated protein 8Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr 1 5 1015 Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His 20 2530 Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 4045 Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 5560 Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 7075 80 Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln 8590 95 Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu100 105 110 Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly AsnVal 115 120 125 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu LysAsn Ile 130 135 140 Lys Glu Phe Leu Asp Ser Phe Val His Ile Val Asp MetPhe Ile Asn 145 150 155 160 Thr Ser 9 23 DNA Artificial Sequencesynthetically generated primer 9 ccaacggcat ggatctcaaa gac 23 10 25 DNAArtificial Sequence synthetically generated primer 10 tcactgtcccagcatcttgt gtttc 25 11 24 DNA Artificial Sequence syntheticallygenerated primer 11 tgcactacag gctccgagat gaac 24 12 25 DNA ArtificialSequence synthetically generated primer 12 catcagaggc aaggaggaaa cacag25

What is claimed is:
 1. A substantially pure mutant IL-15 polypeptide,the polypeptide being: (a) at least 90% identical to wild-type IL-15,and (b) capable of inhibiting at least one of the cellular events thatnormally occurs when wild-type IL-15 specifically binds to a cellsurface receptor.
 2. The mutant polypeptide of claim 1, wherein themutant polypeptide competes with wild-type IL-15 for binding to a cellsurface receptor.
 3. The mutant polypeptide of claim 1, wherein themutant polypeptide binds to a cell surface receptor with at least ashigh an affinity as wild-type IL-15 binds the same cell surfacereceptor.
 4. The mutant polypeptide of claim 1, wherein the mutantpolypeptide binds the cell surface receptor with a Kd of greater than10⁻⁹ M.
 5. The mutant polypeptide of claim 1, wherein at least onemutation is within the domain that binds IL-2Rã.
 6. The mutantpolypeptide of claim 5, wherein the mutation is a deletion, insertion,or substitution of an amino acid residue.
 7. The mutant polypeptide ofclaim 6, wherein the mutation is at position 156 of SEQ ID NO:
 6. 8. Themutant polypeptide of claim 7, further comprising a mutation at position149 of SEQ ID NO:
 6. 9. The mutant polypeptide of claim 7, wherein themutation at position 156 of SEQ ID NO: 6 is a substitution of aspartatefor glutamine.
 10. The mutant polypeptide of claim 8, wherein themutation at position 149 of SEQ ID NO: 6 is a substitution of aspartatefor glutamine.
 11. The mutant polypeptide of claim 1, wherein thepolypeptide is part of a chimeric molecule that comprises a heterologouspolypeptide.
 12. The mutant polypeptide of claim 11, wherein theheterologous polypeptide is the Fc region of IgG.
 13. The polypeptide ofclaim 12, wherein the Fc region of IgG increases the circulatinghalf-life of the mutant polypeptide.
 14. The mutant polypeptide of claim12, wherein the Fc region of IgG is capable of activating the complementlysis system and Fc receptor bearing phagocytes.
 15. The mutantpolypeptide of claim 12, wherein the Fc region of IgG is incapable ofactivating the complement lysis system.
 16. The mutant polypeptide ofclaim 1, further comprising an antigenic tag.
 17. The mutant polypeptideof claim 16, wherein the antigenic tag is a FLAG sequence comprisingAsp-Tyr-Lys-Asp-Asp-Asp-Lys (SEQ ID NO: 4).
 18. An isolated nucleic acidmolecule encoding the polypeptide of claim
 1. 19. A vector comprisingthe nucleic acid molecule of claim
 18. 20. The vector of claim 19,wherein the vector is an expression vector.
 21. A biological cellcomprising the expression vector of claim
 20. 22. A method ofsuppressing the immune response in a patient, the method comprisingadministering to the patient the mutant IL-15 polypeptide of claim 1.23. The method of claim 22, wherein the patient has an autoimmunedisease or is at risk of developing an autoimmune disease.
 24. Themethod of claim 23, wherein the autoimmune disease is a rheumaticdisease selected from the group consisting of systemic lupuserythematosus, Sjögren's syndrome, scleroderma, mixed connective tissuedisease, dermatomyositis, polymyositis, Reiter's syndrome, and Behcet'sdisease.
 25. The method of claim 23, wherein the autoimmune disease isrheumatic arthritis.
 26. The method of claim 23, wherein the autoimmunedisease is type I diabetes.
 27. The method of claim 23, wherein theautoimmune disease is an autoimmune disease of the thyroid selected fromthe group consisting of Hashimoto's thyroiditis and Graves' Disease. 28.The method of claim 23, wherein the autoimmune disease is an autoimmunedisease of the central nervous system selected from the group consistingof multiple sclerosis, myasthenia gravis, and encephalomyelitis.
 29. Themethod of claim 23, wherein the autoimmune disease is a variety ofphemphigus selected from the group consisting of phemphigus vulgaris,phemphigus vegetans, phemphigus foliaceus, Senear-Usher syndrome, andBrazilian phemphigus.
 30. The method of claim 23, wherein the autoimmunedisease is psoriasis.
 31. The method of claim 23, wherein the autoimmunedisease is inflammatory bowel disease.
 32. The method of claim 22,wherein the patient has acquired immune deficiency syndrome (AIDS). 33.The method of claim 22, wherein the patient has received a transplant ofa biological organ, tissue, or cell.
 34. The method of claim 22, whereinthe patient has received a vascular injury.
 35. A method of reducing theviability of a cell that expresses a receptor for IL-15, the methodcomprising exposing the cell to the mutant IL-15 polypeptide of claim 1.36. The method of claim 35, wherein the cell is malignant.
 37. A methodof diagnosing a patient as having a disease or condition that could betreated with a mutant IL-15 polypeptide, the method comprisingdetermining whether a biological sample obtained from the patientcontains cells that are bound by a polypeptide comprising IL-15 and anantigenic tag, the occurrence of binding indicating that the cells canbe bound by mutant IL-15 polypeptide in vivo and thereby inhibited fromproliferating in response to wild-type IL-15 in vivo.
 38. A method ofsuppressing the immune response in a patient, the method comprisingadministering to the patient a polypeptide comprising a mutantinterleukin-15 (IL-15) polypeptide having a mutation at position 149 ofSEQ ID NO: 6 and an Fc region of an IgG molecule that activates thecomplement lysis system.
 39. The method of claim 38, wherein the patienthas an autoimmune disease or is at risk of developing an autoimmunedisease.
 40. The method of claim 39, wherein the autoimmune disease isrheumatoid arthritis.
 41. The method of claim 39, wherein the autoimmunedisease is inflammatory bowel disease.
 42. The method of claim 38,wherein the mutation at position 149 of SEQ ID NO: 6 is a substitutionof aspartate for glutamine.
 43. The method of claim 38, wherein thepolypeptide further comprises a mutation at position 156 of SEQ ID NO:6.
 44. The method of claim 43, wherein the mutation at position 156 ofSEQ ID NO: 6 is a substitution of aspartate for glutamine.
 45. Themethod of claim 38, wherein polypeptide further comprises an antigenictag.
 46. The method of claim 45, whereinthe antigenic tag is a FLAGsequence comprising Asp-Tyr-Lys-Asp-Asp-Asp-Lys (SEQ ID NO: 4).
 47. Amethod of reducing the viability of a cell that expresses a receptor forIL-15, the method comprising exposing the cell to a polypeptidecomprising a mutant interleukin-15 (IL-15) polypeptide having a mutationat position 149 of SEQ ID NO: 6 and an Fc region of an IgG molecule thatactivates the complement lysis system.
 48. The method of claim 47,wherein the cell is malignant.
 49. The method of claim 47, wherein themutation at position 149 of SEQ ID NO: 6 is a substitution of aspartatefor glutamine.
 50. The method of claim 47, wherein the polypeptidefurther comprises a mutation at position 156 of SEQ ID NO:
 6. 51. Themethod of claim 50, wherein the mutation at position 156 of SEQ ID NO: 6is a substitution of aspartate for glutamine.
 52. The method of claim47, wherein polypeptide further comprises an antigenic tag.
 53. Themethod of claim 52, whereinthe antigenic tag is a FLAG sequencecomprising Asp-Tyr-Lys-Asp-Asp-Asp-Lys (SEQ ID NO: 4).